The role of fibronectin in factor VIII/von Willebrand factor cryoprecipitation

To evaluate the role of fibronectin (Fn) in factor VIII (FVIII) and von Willebrand factor (vWf) cryoprecipitation, factor VIII procoagulant activity, factor VIII coagulant antigen, factor VIII-related antigen and von Willebrand ristocetin cofactor activity were measured in cryoprecipitate and cryosupernatant from normal and Fn-depleted plasmas. Following cryoprecipitation of normal plasmas, most of the FVIII and almost all the FvWf recovered were found with a part of Fn and of fibrinogen in cryoprecipitate. Fn-depleted plasmas prepared either by affinity chromatography on gelatin or by immunoadsorption on monoclonal anti-Fn antibodies behaved differently : although their cryoprecipitate contained normal fibrinogen levels, neither FVIII nor FvWf was precipitated. Experiments performed with Fn-depleted plasma to which purified fibronectin had been added, and samples of plasma with decreased Fn levels (0.01 to 0.2 g/l) suggest that there is a relation between initial Fn level and the extent of FVIII/vWf cryoprecipitation. We conclude that Fn, like fibrinogen, is necessary to induce cryoprecipitation of FVIII/vWf and that an initial plasma level of 0.2 g/l is sufficient to obtain good recovery of FVIII/vWf in cryoprecipitate.     

Hormonal control of haemostasis during hypoglycaemia in diabetes mellitus

Factor VIII (FVIII) and plasminogen activator activity (PAA) rise during hypoglycaemia, and this might contribute to the vascular complications of diabetes. Similar changes in haemostasis accompany raised plasma levels of vasopressin (aVP) and adrenaline. To investigate the effects of these hormones on haemostasis during hypoglycaemia and the role of plasma insulin concentrations, eight insulin-dependent diabetic patients underwent controlled hypoglycaemia for 20 min and 13 diabetic patients were investigated during hyperinsulinaemia with blood glucose maintained at 8.0 mmol/l. During hypoglycaemia, insulin levels increased to median values of 114 mU/l, a VP rose from 0.5 to 4.4 (p less than 0.005) pg/ml and adrenaline from 0.4 to 4.4 nmol/l (p less than 0.005). FVIII coagulant activity (FVIII:C) rose from 0.75 to 1.09 IU/ml (p less than 0.01) and the ristocetin co-factor (FVIIIR:Co) and von Willebrand factor antigen (vWF:Ag) showed similar responses. PAA increased from 156 to 745 units (p less than 0.005). During hyperinsulinaemia, insulin rose following infusion from 24 to 52 and 118 mU/l, maintained for an hour at each level. Despite this, plasma aVP, FVIII:C, FVIIIR:Co, vWF:Ag and PAA remained unchanged. This study indicates that the marked changes in FVIII, vWF and PAA concentrations which accompany hypoglycaemia depend on low blood glucose and not raised plasma insulin. The response in probably mediated by increases in adrenaline and aVP, which are part of the physiological response to hypoglycaemia.     

The effect of DDAVP infusion on thrombin generation in platelet-rich plasma of von Willebrand type 1 and in mild haemophilia A patients

In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 microg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases. To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; approximately 40% of vWF is required for half-normal thrombin generation in PRP. It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.     

Effect of exercise, DDAVP, and epinephrine on the factor VIII:C/von Willebrand factor complex in normal dogs and von Willebrand factor deficient Doberman pinscher dogs

Endothelial cells in biopsied blood vessels from von Willebrand factor (vWf)-deficient Doberman pinscher dogs contain immunologically detectable vWf. These dogs and normal dogs were treated with DDAVP (0.6 microgram/kg) and epinephrine (0.5 microgram/kg/min for 30 minutes) and were exercised, using 5 different exercise protocols, (3-4 m/s for 5-40 minutes at 0-5% grade) to determine if treatments reported to increase plasma factor VIII:C/vWf complex in humans would elevate canine plasma vWf. Following the two most strenuous exercise conditions--30 and 40 minutes--plasma von Willebrand factor antigen (vWf:Ag) increased in normal dogs by 30% and 70%, respectively. Factor VIII:C was increased 47% by the most strenuous exercise conditions. The vWf-deficient dogs would not exercise beyond 30 minutes and neither vWf:Ag nor factor VIII:C activity increased. Following DDAVP, plasma vWf:Ag increased in the normal dogs by 47% and factor VIII:C activity was increased by 48%. Factor VIII:C activity increased by 30% in the vWf-deficient dogs, but there was only a slight change in vWf:Ag. Bleeding time decreased in 5 of 6 vWf-deficient dogs. In the normal dogs vWf:Ag increased by 14% after epinephrine infusion, but factor VIII:C activity did not change; neither parameter was altered in the vWf-deficient dogs. While the factor VIII:C/vWf:Ag complex was increased in the normal dog by exercise and DDAVP, the increase is not as pronounced as has been reported for humans. It is not known whether the poor response of the vWf-deficient dog is due to low levels of vWf in their endothelium or to a release defect.     

Effect of controlled hyperglycaemia on factor VIII concentrations in insulin dependent diabetes mellitus

Patients with diabetes mellitus have higher levels of coagulation factor VIII than the non-diabetic population. This may be a result of poor metabolic control and could contribute to the development of microvascular complications. During ketoacidosis there are acute changes in plasma concentrations of coagulation factors, some of which may be mediated by the rise in vasopressin that occurs. We have investigated the effects of hyperglycaemia without ketosis on some aspects of haemostasis by manipulating blood glucose concentrations using a Biostator. After a 1h run-in period with the blood glucose at 5 mmol/l, the blood glucose was maintained at 5, 15 and 25 mmol/l and maintained for one hour at each level in six male patients with insulin-dependent diabetes. Insulin was infused at 0.25 mu/kg/min. Venous blood samples were taken at the beginning and end of each hour after the run-in period for assays of factor VIII coagulant activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), ristocetin co-factor (FVIIIR:Co), activated partial thromboplastin time (APTT) and vasopressin (aVP). There was a slight, though statistically insignificant fall in median factor VIII:C concentration at each incremental level of increase in blood glucose. Values (at the beginning and end of each hour) were: 1.0 and 1.1 iu/ml at 5 mmol/l; 0.95 and 0.79 iu/ml at 15 mmol/l; and 0.74 and 0.84 iu/ml at 25 mmol. vWF:Ag and FVIIIR:Co were unchanged. Plasma aVP fell slightly from 1.1 to 0.5 pg/ml. The results indicate that high levels of FVIII seen in diabetes are not due to short-term increases in blood glucose and that acute hyperglycaemia does not promote pro-coagulant changes in blood.     

Local and systemic effects of intra-arterial desmopressin in healthy volunteers and patients with type 3 von Willebrand disease. Role of interleukin-6

Intra-arterial desmopressin caused dose and time dependent increases (p <0.001 for all) in forearm blood flow (all doses) and plasma tissue plasminogen activator (t-PA) concentrations (desmopressin > or = 70 ng/min). Although plasma t-PA concentrations rose in both forearms, there was a modest local release of t-PA in the infused forearm (14 ng/100 mL of tissue/min, p <0.05). At desmopressin doses > or = 300 ng/min, plasma von Willebrand factor (vWf) and Factor VIII:C concentrations rose in both forearms (p <0.001) and correlated with the rise in interleukin-6 concentrations (r = 0.92, p <0.001: r = 0.85, p = 0.002 respectively). Neither desmopressin nor substance P caused t-PA, vWf or Factor VIII:C release in the patients, although desmopressin increased plasma interleukin-6 concentrations as in healthy volunteers. We conclude that desmopressin releases t-PA, vWf and Factor VIII:C predominantly via systemic mechanisms, possibly mediated by cytokine release. Patients with type 3 vWD appear to have a generalised failure to release t-PA acutely despite a normal interleukin-6 response to desmopressin infusion.     

Standardisation of factor VIII--V. Calibration of the 2nd International Standard for Factor VIII and von Willebrand factor activities in plasma.

The proposed 2nd International Standard for Factor VIII and von Willebrand Factor activities in plasma, NIBSC code 87/718, was assayed against the 1st IRP, 80/511, and against fresh normal plasma, in 21 laboratories. There were no significant differences between the various assay methods for factor VIII antigen, von Willebrand factor antigen, and von Willebrand factor ristocetin co-factor activity. For factor VIII clotting activity there was a significant difference between the results of one-stage and two-stage assays. Plasma 87/718 has now been established by the WHO Expert Committee on Biological Standardisation as the 2nd IS for factor VIII and vWF in plasma with the following potencies: VIII:C 0.60 IU/ampoule; VIII:Ag 0.91 IU/ampoule; vWF:Ag 0.91 IU/ampoule; vWF/RCo 0.84 IU/ampoule.     

Studies on the factor VIII/von Willebrand factor antigen on human platelets

A purified protein having both Factor VIII activity (coagulant assay) and von Willebrand factor activity (platelet retention and ristocetin aggregation assays) was used to immunize a goat. The resulting antiserum neutralized Factor VIII coagulant activity, decreased platelet retention of normal blood and blocked ristocetin aggregation of normal platelet rich plasma.

This same antiserum acted as an “anti-platelet” antibody in serotonin release, platelet aggregation and immunofluorescence with normal, hemophilic and von Willebrand platelets. However, after suitable absorption with small numbers of platelets this antiserum recognized Factor VIII/von Willebrand factor antigen only on normal and hemophilic platelets but not on platelets from two patients with severe von Willebrand's disease.

It is possible that antisera produced against Factor VIII purified from plasma not rendered completely free of platelets contains antibodies to platelet membrane material.     

Plasma-derived biological medicines used to promote haemostasis

Several biological medicines derived from human and animal plasmas can effectively improve haemostasis in individuals with inherited or acquired defects in haemostasis. Factor VIII and factor VIII/vWF and factor IX concentrates are used to treat haemophilia A, von Willebrand disease and hemophilia B respectively. Cryoprecipitates are used to treat hypofibrinogenemia and von Willebrand disease where desmopressin (DDAVP) is ineffective or when plasma-derived factor VIII/vWF concentrates are unavailable. Thrombin-containing topical haemostatic agents and fibrin sealants are used to control perioperative bleeding. Intravenous immunoglobulin has several uses, including management of patients with autoimmune thrombocytopenias and patients with acquired factor VIII deficiency. Similar to most protein-based biological medicines, all the above products can elicit some level of antibody response, with clinical consequences that vary from mild anaphylaxis to loss of product efficacy. An ongoing potential safety concern with any biological medicine derived from blood/plasma is transmission of blood-borne pathogens. This safety concern has lessened significantly in the past decade as a result of the institution of more effective pre- and post-donation screening that tests for potential pathogens, and institution of pathogen reduction strategies to which many plasma-derived biological medicines are now routinely subjected. This article considers the manufacture, standardization, clinical efficacy and adverse event profiles of the plasma-derived biological medicines currently used to promote haemostasis in patients with inherited or acquired functional defects in haemostasis. It also considers approaches employed to minimize infectivity of biological medicines derived from human and animal plasmas and to manage patients who develop antibodies (inhibitors) to clotting factor concentrate infusions.     

Effect of dextran on factor VIII/von Willebrand factor structure and function

Factor VIII/von Willebrand factor was analyzed before and after the infusion of 500 ml of Dextran 70 to normal volunteers. Factor VIII procoagulant activity, factor VIII related antigen and ristocetin cofactor activity showed a significant decrease, reaching after six hours the minimum level, which did not correlate with the hemodilution effect caused by dextran. Ristocetin-induced platelet agglutination (RIPA) in volunteers' platelet-rich plasma (PRP) did not show any significant change between preinfusion time and six hours after the infusion. Multimeric analysis of von Willebrand factor (vWF) showed a progressive decrease of all the multimers which was more pronounced in the largest multimers. No change was seen in the "triplet" structure of vWF. No effect was noticed when dextran was incubated "in vitro" either with PRP or platelet-poor plasma. The modification induced by dextran is close to the pattern seen in subtype Ib von Willebrand's disease.     

The interaction of the factor VIII/von Willebrand factor complex with hematin

Intravenous infusion of hematin, used in the treatment of acute porphyria, induces a decline in the plasma factor VIII/von Willebrand factor complex (VIII/vWF) and thrombocytopenia. We investigated this problem by studying the interaction between hematin, purified VIII/vWF, and platelets in vitro. Hematin was labeled with either 59Fe or 3H and characterized by gel chromatography. Hematin self-aggregated, forming a complex with an average molecular weight of approximately 10,000 daltons. When incubated with VIII/vWF for 30 min at 37 degrees C and applied to Sepharose CL-4B, the hematin eluted with the VIII/vWF in the void volume. Hematin inhibited the dissociation of factor VIII antigen (VIII:Ag) from the von Willebrand antigen (vWF:Ag) in 0.25 M CaCl2, and reversed the aggregation of VIII:Ag induced by 0.1 M 6-aminocaproic acid. Both hematin and the hematin-VIII/vWF complex bound to washed normal platelets and to platelets from a patient with Bernard-Soulier syndrome. Thrombasthenic platelets were not aggregatable by hematin, and bound significantly less hematin-VIII/vWF than normal platelets suggesting that hematin-induced platelet activation was required for binding. Likewise, binding was inhibited by PGE1 which also prevented aggregation. We conclude that hematin forms complexes with VIII/vWF, alters the functional activity and dissociation of this compound, and participates in the binding of VIII/vWF to platelets.     

The type of factor VIII deficient plasma used influences the performance of the Nijmegen modification of the Bethesda assay for factor VIII inhibitors.

We have investigated the influence of the type of factor VIII deficient plasma used on the assay results of the Nijmegen modification of the Bethesda method for factor VIII inhibitors. Immuno depleted factor VIII deficient plasmas, lacking besides factor VIII also von Willebrand factor, gave decreased inhibitor titres compared to assay results with factor VIII deficient plasmas containing von Willebrand factor suggesting the need of the latter in the test system for the stability of factor VIII:C. Moreover the performance of the assay with immuno depleted plasma was contaminated in a certain type of this plasma by the presence of a factor VIII:C inhibitor. Chemically depleted factor VIII deficient plasma appeared to give falsely elevated titres when used in combination with other types of deficient plasmas as substrate plasma in the factor VIII:C assay due to the presence of activated factor Va in the preparation. Suggestions are described with respect to the observed limitations in order to obtain reliable results.     

Variations in coagulation factors in women: effects of age, ethnicity, menstrual cycle and combined oral contraceptive

To assess variations of coagulation factors in women, 123 women were included in a cross-sectional study of the effect of age, ethnic origin, blood group and menstrual cycle on surface induced coagulation time (activated partial thromboplastin time) and plasma levels of Factor VIII clotting assay, von Willebrand factor antigen, von Willebrand factor activity and factor XI. The effect of menstrual cycle was further assessed in a longitudinal study including 39 Caucasian women, 20 of whom were using combined oral contraceptives. Activated partial thromboplastin time was longer in women with blood groups B or O, and plasma levels of factor VIII clotting assay, von Willebrand factor antigen and von Willebrand factor activity were significantly higher in black women. Fibrinogen, von Willebrand factor antigen and von Willebrand factor activity concentrations showed strong cyclic variations with peak values in the luteal phase. This pattern was dampened for von Willebrand factor antigen and von Willebrand factor activity but completely disappeared for fibrinogen with the use of combined oral contraceptives. There was a cyclical pattern for factor VIII clotting assay in pill users, evidence of which was not evident in non-pill users. There were strong associations between the levels of von Willebrand factor antigen and von Willebrand factor activity and age, with levels rising by an average of 0.17 and 0.15 U/ml, respectively, for each 10 year increase in age. In conclusion, there are great inter- and intraindividual variations in coagulation markers in women due to different physiological conditions such as age, ethnicity, blood group and phases of the menstrual cycle. However, there were no significant associations between coagulation markers and weight, alcohol consumption or smoking status.     

Purification of the factor VIII complex

Two high purity factor VIII concentrates, type I and type II were developed for clinical trials in patients with hemophilia A and von Willebrand's disease. Fresh frozen plasma containing 1% polyethylene glycol 4000 was thawed to form cryoprecipitate, which was subsequently dissolved in citrate buffer. By addition of glycine buffer to a final concentration of 2.0 M at 26°C, the bulk of fibrinogen was precipitated while factor VIII remained in solution. Factor VIII was precipitated from the glycine supernatant by addition of solid sodium chloride. The recovery of factor VIII procoagulant activity (VIII:C) per kg plasma was 271±23 units (n=4) and 386±47 units (n=7) for the type I and the type II preparations, respectively, while the recovery of von Willebrand factor related activity (ristocetin cofactor, VIIIR:RC) was 518±75 units and 718±90 units per kg plasma, respectively. The specific activity (units per mg protein) of VIII:C in the type I and type II preparations were 2.53±1.02 and 7.56±1.33, respectively. The specific activity (units per mg protein) of VIIIR: RC for the type I and type II preparations were 4.86±2.32 and 13.6±3.7, respectively. VIIIR:Ag was present as multimers in both preparations, and the multimeric pattern was similar to that of normal plasma. The preparations have the ability to correct the prolonged bleeding time in severe von Willebrand's disease. The factor VIII complex in the type II preparations was further purified by gel filtration on Sephacryl S-1000. This preparation was free of fibrinogen and fibronectin. Its specific activity in terms of VIII:C was 47 u/mg protein and 104 u/mg protein in terms of VIIIR:RC. The subunit of reduced factor VIIIR:Ag had M_r of 210 Kd on sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Von Willebrand's disease associated with thrombocytopenia and a fast migrating factor VIII related antigen.

Five cases of an atypical von Willebrand syndrome are described in a family with a bleeding diathesis transmitted in an autosomal dominant mode for three generations. The five patients had an intermittent mild thrombocytopenia, a prolonged bleeding time regardless of the platelet count and an abnormal in vivo and in vitro platelet adhesiveness. Clot retraction, platelet factor-3 availability, ristocetin, ADP, epinephrine and collagen induced platelet aggregations were normal. In all five subjects, Factor VIII activity (VIII:C) was moderately decreased (18–34%), Factor VIII related antigen (VIII:Ag) (54–256%) was higher than VIII:C and the von Willebrand factor (VIII:WF) measured by paraformaldehyde fixed platelet ristocetin induced macroscopic aggregation was decreased. These patients had clinically very little bleeding problem and one of them responded like a classic von Willebrand to fresh frozen plasma given for surgery. They all had an VIII:Ag which migrated faster than the normal control towards the anode in crossed-immunoelectrophoresis. In one patient studied the relative anodal migration of VIII:Ag varied in time; a decrease in anodal migration corresponded to a decrease in VIII:Ag but did not correlate with any other parameter of factor VIII or platelet function.     

Platelet adhesion to collagen in subtypes of type I von Willebrand's disease is dependent on platelet von Willebrand factor

Von Willebrand's disease type I, characterized by low levels of factor VIII coagulant activity (VIII: C), von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (RiCof) (1), can be subdivided on the basis of platelet von Willebrand factor into subtype platelet normal, platelet discordant, and platelet low (2). We have investigated the contribution of platelet von Willebrand factor in these various subtypes to platelet adhesion using the rectangular perfusion chamber of Sakariassen et al. (3) with fibrillar collagen or a fibroblast matrix as adhesive surfaces. Platelet adhesion to fibrillar collagen was decreased in all subtypes of von Willebrand's disease, but not as low as in severe von Willebrand's disease. A close correlation was observed between platelet adhesion to collagen and plasma vWF:Ag in severe von Willebrand's disease, subtype platelet low, subtype platelet discordant, and normal controls. The platelet adhesion in subtype platelet normal was higher than expected from the plasma vWF:Ag level. Perfusions in which washed platelets were added to a human albumin solution together with red blood cells gave similar adhesion values in subtype platelet normal and normal controls; adhesion was decreased in subtype platelet discordant, and the lowest values were found in subtype platelet low and in severe von Willebrand's disease. These data indicate that platelet von Willebrand factor may contribute to platelet adhesion, when plasma von Willebrand factor is low. Perfusion studies over a fibroblast matrix gave similar low adhesion values for subtype platelet low and platelet normal, indicating that the contribution of platelet von Willebrand factor can only be observed on a strongly activating surface such as fibrillar collagen.     

Effects of toll-like receptor-4 gene polymorphisms on soluble P-selectin and von Willebrand factor levels in hypercholesterolemic patients

Toll-like receptor-4 (TLR-4) gene polymorphisms have been associated with a lower risk of atherosclerosis. High levels of soluble P-selectin (sP-selectin) and von Willebrand factor predict an increased risk for cardiovascular events and correlate to atherosclerotic risk factors. The relationship between these markers and TLR-4 gene polymorphisms was evaluated in a cohort of consecutive hypercholesterolemic outpatients. TLR-4 gene polymorphisms were detected in 48 out of 330 (14%) patients with hypercholesterolemia. Lipid and inflammatory markers, sP-selectin and von Willebrand were evaluated in carriers and in 96 (ratio 2:1 to cases) age- and sex-matched TLR-4 wild-type patients randomly selected from the same population. A cohort of normocholesterolemic outpatients (n = 262) served as the control group. sP-selectin was sensibly lower in carriers of TLR-4 variants as compared to wild-types and controls (89 ng/ml vs. 162 ng/ml and 163 ng/dl, respectively, p = 0.0001). Similarly, carriers showed lower von Willebrand factor values (683 mU/ml) than wild-types (910 mU/ml; p = 0.001). In multivariate analysis, TLR-4 gene polymorphisms were positively associated with sP-selectin, whereas the relationship with von Willebrand factor was no longer significant. HMG-CoA reductase inhibitors reduced sP-selectin and von Willebrand factor levels independently of TLR-4 gene variants. Plasma concentrations of these markers, however, remained lower in carriers of TLR-4 gene polymorphisms even after cholesterol lowering. In conclusion, carriership of Asp299 and Thr399Ile TLR-4 gene polymorphisms is associated with lower levels of sP-selectin and von Willebrand factor among hypercholesterolemic patients. While the underlying mechanisms remain to be investigated, such an association may indicate a protective effect of TLR-4 variants for atherosclerosis.     

Von Willebrand factor/factor VIII adsorption to surfaces from human plasma

The adsorption of human von Willebrand factor/factor VIII (VWF/VIII) from citrated plasma to a series of polymeric surfaces and glass was measured with both prelabelled ¹²⁵I VWF/VIII and an ¹²⁵I antibody to VWF/VIII. Adsorption to the various surfaces differed but was generally quite low (0.24 to 6.5 ng/cm²). Results from both techniques agreed well. The relationship between VWF/VIII adsorption and the thrombogenicity of the surfaces was examined but no clear trend could be discerned. Further studies will therefore be required to determine whether the variation in VWF/VIII adsorption influences the thrombogenicity of the surfaces.     

The dynamic inter-relationship between factor VIII and von Willebrand factor.

A monospecific heterologous antibody which specifically inhibited von Willebrand factor (W.F.) but had no factor VIII procoagulant (VIIIc) neutralising properties was insolubilised by attachement to sepharose beads. When fresh normal plasma was mixed with such beads, VIIIc, W.F. and factor VIII related antigen (VIIIAg) were removed in equal proportions, with the VIIIc lost from the plasma being detectable attached to the sepharose. This VIIIc was removed from the sepharose and largely recovered in the eluate after treatment with 0.2M calcium chloride. Subsequent addition of plasma resulted in a high transfer of VIIIc from the plasma onto the sepharose beads without accompanying W.F. and VIIIAg. Similar experiments, with initial treatment of antibody attached sepharose beads with serum, resulted in no independent segregation of VIIIc on subsequent addition of plasma. It is suggested that VIIIc and W.F. exist in plasma as separate, but attached molecules in a dynamic equilibrium. In serum, W.F. loses its ability to attach VIIIc.     

High levels of plasma FVIII and vWF in the toxic epidemic syndrome patients

Factor VIII and von Willebrand factor proteins were evaluated in 115 patients having the chronic phase of the Toxic Epidemic Syndrome (TES), a new multisystemic disease probably caused by the ingestion of denatured rapeseed oil, and in 50 control volunteers. Higher circulating levels of factor VIII procoagulant activity (VIII:C) (158 +/- 58.4 U/dl), von Willebrand factor antigen (vWF:Ag) (166.1 +/- 55.5 U/dl) and von Willebrand factor ristocetin cofactor activity (vWF:RCo) (178.7 +/- 55.2 U/dl) were seen in TES patients (p less than 0.001, TES patients versus control subjects, for each parameter). The increased levels of vWF:Ag and vWF:RCo observed in TES patients correlated with the scleroderma like lesion of the skin, with the sicca syndrome and with Raynaud's phenomenon (p less than 0.01), but not with other clinical manifestations. The multimeric analysis of vWF in 92% of the TES patients was similar to that found in normal plasma, but in the remaining 8% a very slight increase of larger vWF multimers in plasma were observed. The raised levels of vWF found in TES patients in the chronic phase may reflect an "in vivo" vascular injury.