Possible effect of secretor locus on plasma concentration of factor VIII and von Willebrand factor

A significant fraction (30%) of the genetically determined variance in plasma concentration of the von Willebrand factor antigen (vWf:Ag) has been shown to be related to ABH determinants. Individuals with blood group O, who have the highest amounts of blood group H substance, have the lowest concentration of vWf:Ag. The Lewis substances, Le(a) and Le(b), are biochemically closely related to the ABH substances as both can be produced from the same precursor substance. We studied the effect of the presence of the Lewis antigens on the plasma concentration of vWf:Ag and factor VIII antigen (VIII:Ag) in 323 individuals of different ABO groups from a series of twins and in 58 blood donors of blood group O. Among persons belonging to blood group O, those with the Le(a) antigen had a higher concentration of both vWf:Ag and VIII:Ag than individuals lacking Le(a). Le(a+b-) people are nonsecretors and Le(a-b+) people are secretors of ABH substance. Thus, the lowest concentration of vWf:Ag and VIII:Ag was found in group O secretors. The effect is most likely due to an effect of the secretor locus. This finding may be of importance for the detection of carriers of hemophilia A and for the diagnosis of type I von Willebrand disease.     

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.     

Correlation between von Willebrand factor antigen,von Willebrand factor ristocetin cofactor activity and factor VIII activity in plasma

Background The laboratory diagnosis of von Willebrand Factor (VWF) deficiencies includes qualitative and quantitative measurements of VWF and clotting factor VIII (FVIII). Since the FVIII activity is frequently normal in patients with mild type 1 or 2 von Willebrand disease (VWD), there is controversy whether FVIII testing should accompany VWF Antigen (VWF:Ag) assay. Methods The aim of this study was to explore the correlation between VWF:Ag, VWF ristocetin cofactor activity (VWF:RCo) and FVIII in 213 consecutive patients undergoing screening for VWD. Results Forty-six patients were identified with VWF:Ag levels lower than the diagnostic threshold (54 IU/dl). A significant correlation was observed between VWF:Ag and VWF:RCo (r = 0.892; p < 0.001), VWF:Ag and FVIII (r = 0.834; p < 0.001), VWF:RCo and FVIII (r = 0.758; p < 0.001). Receiver operating characteristic curve analysis of the VWF:Ag assay revealed an area under the curve of 0.978 and 0.957 for detecting life-threatening values of FVIII (<30 IU/dl) and VWF:RCo (<40 IU/dl), respectively. The negative and positive predictive values at the VWF:Ag threshold value of 54 IU/dl were 100% and 33% for detecting life-threatening FVIII deficiencies, 94% and 80% for identifying abnormal values of VWF:RCo. Conclusions Due to the excellent correlation between VWF:Ag and FVIII and to the diagnostic efficiency of VWF:Ag for identifying abnormal FVIII levels in patients with VWF deficiency, routine measurement of FVIII may not be necessary in the initial screening of patients with suspected VWD. However, the limited negative predictive value of VWF:Ag for identifying type 2 VWD does not allow to eliminate VWF:RCo or VWF:FVIIIB assays from the diagnostic workout.     

The complex multimeric composition of factor VIII/von Willebrand factor

We have analyzed the multimeric structure of factor VIII/von Willebrand factor in plasma by sodium dodecyl sulfate electrophoresis using gels of varying porosity and a discontinuous buffer system. Factor VIII/von Willebrand factor bands were identified by reaction with 125I-labeled affinity-purified antibody and subsequent autoradiography. In 1% agarose gels, normal plasma displayed a series of sharply defined oligomers. However, increasing the agarose concentration to 2.0% or utilizing mixtures of 0.8% agarose--1.75% acrylamide revealed two bands of lesser intensity interposed between the major bands. When the acrylamide concentration in the gels was increased to 2.5%, bands with a faster mobility than IgM and fibronectin were now evident. Type IIA von Willebrand's disease showed not only an absence of the larger multimers but also a relative increase in several of the newly identified bands as compared to type IIB, type I, and normal. These studies suggest that factor VII/von Willebrand factor in IIA von Willebrand's disease is structurally different from that in other forms of the disorder. They also indicate that the multimeric composition of factor VII/von Willebrand factor is more complex than can be explained by simple linear polymerization of a single protomer.     

Increased factor VIII/von Willebrand factor antigen and von Willebrand factor activity in renal failure.

Factor VIII/von Willebrand factor antigen and von Willebrand factor activity (ristocetin assay) were studied in 12 patients in renal failure. A dramatic increase in both activities was observed (antigen 315 +/- 30 per cent in patients verus 104 +/- 9 per cent in control subjects; activity 402 +/- 48 per cent in patients versus 111 +/- 5 per cent in control subjects; p less than 0.001 for both). Since von Willebrand factor is thought to play at least a facilitative role in the development of arteriosclerosis, these increased activities may contribute to the premature arteriosclerosis reported in patients with chronic renal failure undergoing dialysis.     

Comparative analysis and classification of von Willebrand factor/factor VIII concentrates: impact on treatment of patients with von Willebrand disease

von Willebrand disease (vWD) is a bleeding disorder that results from defects in the quality or quantity of von Willebrand factor (vWF), a glycoprotein essential for normal thrombus formation. vWF circulates in plasma as multimers in sizes ranging up to 20,000 kd. The high molecular weight vWF (HMWvWF) multimers are most essential for primary hemostasis, whereas the lower molecular weight multimers are less functionally active. For many patients, the treatment of choice is factor replacement with a vWF/FVIII concentrate, preferably one with a high content of HMWvWF multimers. Given that the commercially available vWF/FVIII concentrates seem to differ substantially in their biochemical properties as well as in their clinical efficacy, we did a comparative study with 12 vWF/FVIII concentrates to investigate content and activities of FVIII and vWF, as well as the content of HMWvWF multimers. The content of HMWvWF multimers varied considerably among the 12 concentrates. The specific vWF activities, as assessed by ristocetin cofactor activity (vWF:RCo) and collagen-binding activity (vWF:CB), correlated well with the HMWvWF content of the products. Of the products tested, Haemate P/Humate-P had the highest content of HMWvWF multimers (with a multimer pattern closest to that of normal human plasma), the highest specific vWF activities, and the highest values of vWF:RCo and vWF:CB per unit of FVIII:coagulant (C). The goal of bleeding prophylaxis and treatment in type 2, severe type 1, and type 3 vWD patients is to normalize vWF activities (vWF:RCo and vWF:CB) and FVIII:C preferentially by vWF/FVIII concentrates containing the high vWF multimers and a high vWF:RCo/FVIII ratio to achieve normal primary and secondary hemostasis. Based on the present study of a comparative analysis of currently available vWF/FVIII concentrates, a classification of vWF/FVIII products is proposed.     

An electroblotting technique for the detection of factor VIII/von Willebrand factor multimers in plasma

A new, relatively simple technique has been developed in order to study the multimers of factor VIII/von Willebrand factor (VIII/vWF). It involves electrophoresis on SDS agarose gels and electrophoretic transfer (electroblotting) of the separated protein bands onto nitrocellulose membranes, to which they are non-covalently bound. VIII/vWF multimers are then detected by 125I-labelled antibodies to VIII/vWF, and autoradiography. Optimum electrophoretic transfer occurred at 0.5 A, for 18 h, on 0.8% agarose gels, thus enabling detection of the multimeric profile of VIII/vWF in 5 microliters of normal plasma. The multimeric profile for haemophilia A patients was identical to that for normal plasma. In plasma from patients with von Willebrand's disease (vWd), various patterns were seen, with a preponderance of smaller multimers in type II (atypical) vWd, similar to those seen in cryosupernatant. Heterogeneity within a particular type of vWd was also evident. Investigation of commercial factor VIII concentrates showed the presence of 'doublets' of VIII/vWF. Unlike other reported techniques, the rapid transfer and fixing of the protein bands to the nitrocellulose, minimizes loss of resolution, and handling of the paper is easier. It is possible to cut a sample electrophoresis strip into several areas, for incubation with different antibodies. Preliminary experiments also suggest that double antibody techniques are possible, or even removal of a first radiolabelled antibody by low pH, and then incubation of the separated proteins with a second, unrelated antibody.     

Relationship between ABO and Secretor genotype with plasma levels of factor VIII and von Willebrand factor in thrombosis patients and control individuals

In contrast to earlier reports, this study examined the relationship between plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) and ABO blood group and secretor status at the genetic level in 355 patients with venous thrombosis as well as in 236 controls. ABO glycosyl transferase alleles A(1) and B were more frequent in the thrombosis collective and alleles O(1), O(2) and A(2) were more frequent in the controls. A low-risk group for venous thrombosis of individuals with genotypes O(1)O(1), O(1)O(2) and O(1)A(2) (H-antigen rich) could be distinguished from a high-risk group with genotypes A(1)A(1), A(1)B, O(1)A(1) and O(1)B (H-antigen poor). In both the thrombosis and control groups, the H-antigen rich group showed significantly lower levels of FVIII coagulant activity (FVIII:C) and VWF antigen (VWF:Ag) than the H-antigen poor group. The frequency of the different secretor genotypes in the thrombosis group was not different from that in the control group. No significant differences of FVIII:C and VWF:Ag levels were seen between SeSe, Sese and sese individuals in the thrombosis and in the control group. Thus the risk of venous thrombosis is associated with the ABO blood group genotype but not with secretor status.     

The factor VIII/von Willebrand factor complex: basic and clinical issues

Factor VIIII (FVIII) and von Willebrand factor (VWF) are two distinct but related glycoproteins that circulate in plasma as a tightly bound complex (FVIII/VWF). Their deficiencies or structural defects are responsible for the most common inherited bleeding disorders, namely hemophilia A (HA) and von Willebrand's disease (VWD). The VWF has a dual role in hemostasis: first it promotes platelet adhesion to thrombogenic surfaces as well as platelet-to-platelet cohesion during thrombus formation; second, it is the carrier for FVIII in plasma. FVIII acts as a co-factor to accelerate the activation of factor X by activated factor IX in the coagulation cascade. After many years of investigations, the molecular mechanisms of FVIII/VWF interactions are now well known and recent biochemical investigations have confirmed that VWF is a key partner for FVIII, playing significant roles in FVIII function, its production and its stabilization, in its conformation and immunogenicity. FVIII and VWF are both present in most plasma-derived FVIII/VWF concentrates used in clinical practice. FVIII/VWF concentrates can be classified into three main categories according to the degree of their purification. Intermediate-high purity plasma-derived concentrates containing FVIII/VWF currently in use since 1987 carry a low risk of transmitting blood-borne infections. Concentrate safety depends on the interaction of two factors: the decrease of viral plasma load and the increase of viral inactivation. These FVIII/VWF concentrates are currently used in type 3 VWD and in type 1 or 2 VWD patients who are unresponsive to desmopressin (DDAVP). More recently the presence of the physiologic FVIII/VWF complex has been considered to play an important role also in replacement therapy for patients with HA. The correct use of FVIII/VWF concentrates in VWD and HA have been reported in several national and international guidelines.     

Heat-treated factor VIII/von Willebrand factor concentrate in platelet-type von Willebrand's disease

Cryoprecipitate has proved to correct the hemostatic defects in von Willebrand's disease (vWD) and platelet-type vWD. However, recent studies have revealed that transmission of the AIDS retrovirus (HIV) occurs through exposure to blood products including cryoprecipitate. Treatment with heat-treated factor VIII/von Willebrand factor (vWf) concentrates may have certain advantages over treatment with nonheated products, if these preparations are efficacious in these disorders. We found that a commercially available factor VIII/vWf concentrate, Haemate P, contained the high-molecular-weight multimers of vWf and had a ratio of ristocetin cofactor (RCof) to vWf antigen (vWf:Ag) close to unity. In addition, its capacity to directly induce aggregation of platelet-type vWD platelets in vitro was similar to that for cryoprecipitate. When infused into a patient with platelet-type vWD, Haemate P shortened the prolonged bleeding time and caused spontaneous platelet aggregation in vitro with a mild diminution of platelet count. These results indicate that some of the heat-treated factor VIII/vWf concentrates may provide a safer, yet still effective, treatment for platelet-type vWD.     

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.     

Pharmacokinetics and hemostatic effect of different factor VIII/von Willebrand factor concentrates in von Willebrand's disease type III

Summary Four different plasma-derived concentrates composed of coagulation factor VIII (FVIII) and von Willebrand factor (vWF) of varying quality (Hemate-P, Behring; Profilate, Alpha; and FVIII-VHP-vWF, C.R.T.S Lille), or almost purified vWF (Facteur Willebrand, C.R.T.S Lille) and one recombinant FVIII concentrate (Recombinate, Baxter) were given, in doses of 30–60 IU VIII:C/kg or 70–110 IU RCof/kg, to five patients with von Willebrand's disease type III, in order to evaluate the role of the vWF in factor FVIII concentrates. All plasma concentrates except Profilate had a multimeric vWF pattern almost similar to that of normal plasma. Bleeding time (b.t.), VIII:C, vWF: Ag, ristocetin cofactor activity, and multimeric pattern of the plasma-vWF were followed for 72 h. Both Duke b.t. and the multimeric pattern in plasma normalized after infusion of Hemate-P, FVIII-VHP-vWF, and Facteur Willebrand and, to a lesser extent, after Profilate. As expected, in response to Recombinate there was no effect on primary hemostasis, and the half-life of FVIII procoagulant activity (VIII:C) was very short. Normalization of the vWF is important not only for improving the primary hemostasis, but also for maintaining the plasma FVIII concentration on a high level, both by reducing the elimination rate of infused FVIII and via a secondary release of endogenous FVIII. If a prompt hemostatic effect is required, we recommend a concentrate containing both FVIII and all vWF multimers, but for prophylactic treatment, pure vWF may be used.     

Structure and function of the factor VIII/von Willebrand factor complex

In the blood plasma factor VIII is bound to the von Willebrand factor. The primary structure of the two proteins were clarified by gene clonation. Factor VIII descends from a precursor protein with 2,351 amino acids by splitting of 19 amino acid residues and is activated by partial proteolysis. In the blood coagulation factor VIII acts as co-factor for the activation of factor X by factor IX in the presence of phospholipids and Ca++ within the intrinsic coagulation system. The formation of the von Willebrand factor takes place by splitting of 22 and 741 amino acid residues, respectively, from pre-pro-von Willebrand factor via pro-von Willebrand factor. The subunits of the von Willebrand factor consist od 2,050 amino acid residues. In the blood plasma the von Willebrand factor is existing as a mixture of multimeres. Receptors of the von Willebrand factor on the thrombocytic membrane are the glycoproteins GPIb and GPIIb/GPIIIa, by means of which the adhesion of thrombocytes at the subendoethelium of the vascular wall and the aggregation of thrombocytes are mediated.     

Characterization of von Willebrand factor in factor VIII concentrates

Commercial concentrates of factor VIII (FVIII) were analyzed in order to 1) determine the effects of viral inactivation on von Willebrand factor (vWF); 2) evaluate the vWF content of the new, immunopurified concentrates; and 3) assess their potential for correcting the long bleeding time of von Willebrand disease (vWD). Included in our study were products that had been treated to inactivate viruses; older, untreated products; and the new, immunopurified concentrates. We measured von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity (RCoF), and vWF multimeric and subunit composition. A newly developed radioimmunoassay (RIA) was used to quantitate vWF:Ag. The vWF:Ag content varied from 0.083 micrograms/IU FVIII:C for Hemofil M to 32.2 micrograms/IU FVIII:C for Humate-P, whereas pooled normal human plasma (NHP) contained 6.3 micrograms/IU FVIII:C. The RCoF varied from 0.0007 to 2.09 U/IU FVIII:C, with the immunopurified concentrates having the lowest values and Humate-P the highest. The ratio of RCoF to vWF:Ag ranged from 11 to 96 U/mg, as compared to a ratio of 160 for NHP. All of the concentrates lacked the largest vWF multimers, and all had abnormal triplet patterns. Modest differences between some untreated concentrates and their treated counterparts were noted. As expected, the immunopurified concentrates had much lower levels of all vWF activities than the conventionally prepared products. Our data suggest that none of the concentrates have as great a capacity as NHP to correct the prolonged bleeding time of von Willebrand disease.     

The role of plasma-derived factor VIII/von Willebrand factor concentrates in the treatment of hemophilia A patients

Besides preventing bleeding episodes, common goals of the treatment of hemophilia include integrating of patients into a normal social life and optimizing their quality of life. Sufficient amounts of factor VIII (FVIII) concentrates, whether recombinant or plasma-derived, are continuously needed. Guidelines for quality assurance of treatment will be a cornerstone to maintain optimal clinical management of patients especially considering financial aspects. Advances in manufacturing technologies have made possible general availability of modern concentrates for the management of hemophilia A patients. Safety, cost and continuous supply of concentrates must be considered when deciding on a product for replacement therapy. As todays' products have reached an excellent margin of safety with regard to virus transmission, the development and treatment of inhibitors is currently the main concern for physicians and patients. The incidence of inhibitors is influenced by various patient-related factors such as mutation type or severity of the disease. Plasma-derived FVIII concentrates containing von Willebrand factor (VWF) may have clinical advantages over pure FVIII concentrates with regard to inhibitor development and inhibitor eradication. Clinical trials comparing FVIII/VWF concentrates with pure FVIII concentrates are lacking, thus a lower inhibitor incidence has not yet been proven. Data from Germany on immune tolerance induction with FVIII/VWF concentrates indicate higher success rates with these than with pure FVIII concentrates. In addition FVIII/VWF concentrates are the therapy of choice when immune tolerance therapy with pure FVIII products is not successful.     

Characterization of factor VIII/von Willebrand factor concentrates using a modified method of von Willebrand factor multimer analysis

In order to provide patients with von Willebrand disease a factor VIII (FVIII)/von Willebrand factor (vWF) concentrate of reproducible quality, an SDS-agarose gel electrophoresis method has been established to determine the content of the high molecular weight multimers (band 11 and higher) of vWF. This method has been used to characterize the content of high molecular weight vWF multimers in Humate^® P/Haemate^® P, a commercial FVIII/vWF concentrate. The average content of high molecular weight vWF multimers of 47 batches of Humate^® P/Haemate^® P has been determined to be 84.1% of the corresponding bands in normal human plasma. Use of this multimer analysis method for the characterization of five further commercial products revealed clear differences with respect to the high molecular weight vWF multimer content. Furthermore, there is a linear correlation ( r ² = 0.73) between the content of high molecular weight vWF multimers and the specific activity of vWF (determined as vWF:RCoF/vWF:Ag). The method described here for analysis of the content of high molecular weight vWF multimers is a reliable and reproducible method to characterize this class of factor concentrates with respect to vWF multimer composition.     

The von Willebrand factor A-1 domain binding aptamer BT200 elevates plasma levels of von Willebrand factor and factor VIII: a first-in-human trial

von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary hemostasis and clotting, respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomized, placebo-controlled, double-blind trial tested the hypothesis that BT200 is well tolerated and has favorable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: a single ascending dose of BT200 (0.18-48 mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterized, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin-induced aggregation, platelet function under high shear rates, and thrombin generation. The mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dose-dependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagen-adenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (P<0.001), without increasing VWF propeptide levels, indicating decreased VWF/FVIII clearance. This, in turn, increased thrombin generation and accelerated clotting. Desmopressin-induced VWF/FVIII release had additive effects on a background of BT200. Tolerability and safety were generally good, but exaggerated pharmacology was seen at saturating doses. This trial identified a novel mechanism of action for BT200: BT200 dose-dependently increases VWF/FVIII by prolonging half-life at doses well below those which inhibit VWF-mediated platelet function. This novel property can be exploited therapeutically to enhance hemostasis in congenital bleeding disorders.     

Abnormal VIII: von Willebrand factor patterns in the plasma of patients with the hemolytic-uremic syndrome

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.     

Influence of single nucleotide polymorphisms in factor VIII and von Willebrand factor genes on plasma factor VIII activity: the ARIC Study

Factor VIII (FVIII) functions as a cofactor for factor IXa in the contact coagulation pathway and circulates in a protective complex with von Willebrand factor (VWF). Plasma FVIII activity is strongly influenced by environmental and genetic factors through VWF-dependent and -independent mechanisms. Single nucleotide polymorphisms (SNPs) of the coding and promoter sequence in the FVIII gene have been extensively studied for effects on FVIII synthesis, secretion, and activity, but impacts of non-disease-causing intronic SNPs remain largely unknown. We analyzed FVIII SNPs and FVIII activity in 10,434 healthy Americans of European (EA) or African (AA) descent in the Atherosclerosis Risk in Communities (ARIC) study. Among covariates, age, race, diabetes, and ABO contributed 2.2%, 3.5%, 4%, and 10.7% to FVIII intersubject variation, respectively. Four intronic FVIII SNPs associated with FVIII activity and 8 with FVIII-VWF ratio in a sex- and race-dependent manner. The FVIII haplotypes AT and GCTTTT also associated with FVIII activity. Seven VWF SNPs were associated with FVIII activity in EA subjects, but no FVIII SNPs were associated with VWF Ag. These data demonstrate that intronic SNPs could directly or indirectly influence intersubject variation of FVIII activity. Further investigation may reveal novel mechanisms of regulating FVIII expression and activity.