Investigation of plasma von Willebrand factor and circulating platelet aggregating activity in mitomycin C-related hemolytic-uremic syndrome

We have studied the ability of the plasma to induce aggregation of both homologous and heterologous platelets in four patients with hemolytic-uremic syndrome (HUS) associated with chemotherapy with mitomycin C (MMC). Neither platelet aggregation was elicited by patients' plasmas nor the in vitro addition of purified von Willebrand factor (vWF) had any effect on the aggregation pattern. In addition, ristocetin-induced binding of patients' vWF to formaldehyde-fixed platelets was normal, and multimeric vWF analysis revealed a normal structure of patients' plasmatic vWF whatever the clinical stage in which it was studied. These findings suggest that, in spite of the existence of common clinical and biological features in the various forms of HUS, the pathogenesis of MMC-related HUS may be, at least in part, different from that of the other forms of HUS in which both platelet-aggregating activity and alterations in the vWF are found.     

Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.     

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.     

Abnormal platelet von Willebrand factor interaction in patients with TTP

Thrombotic thrombocytopenic purpura (TTP) is associated with abnormal platelet function and disturbances in coagulation; however, a specific causative factor is not defined. Plasma infusion or plasma exchange (PE) are thought to be of benefit in replacing a deficient plasma component or removing some toxic compound. In three patients with TTP, samples taken prior to initiation of PE showed high levels of vWF:Ag in the plasma (208, 264, and 321 U/dl), whereas the VIII:C levels were normal. The vWF:Ag multimer patterns of the plasma demonstrated a decrease in the amount of high molecular weight (HMW) forms. Analysis of the platelets from one patient also showed an increase in the HMW multimers. Platelets from all three patients showed a decreased ability to absorb vWF:Ag, with little or no absorption of the HMW forms. Following extensive PE and resolution of disease, the platelets regained their ability to absorb vWF:Ag in the one patient examined.     

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.     

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.     

Function of von Willebrand factor in children with diarrhea-associated hemolytic-uremic syndrome (D+ HUS).

Reports on von Willebrand factor (vWF) in hemolytic-uremic syndrome (HUS) are not unequivocal. Because of potential pathogenic implications, we examined the ability of vWF to bind to collagen in vitro, which reflects its function. Plasma vWF antigen (vWF:Ag) and collagen-binding activity (vWF:CBA) were measured by enzyme-linked immunosorbent assay in children with (1) diarrhea-associated (D+) HUS (n = 27), (2) chronic renal insufficiency (CRI) (n = 8), (3) gastroenteritis (GE) not associated with HUS (n = 15), (4) immune thrombocytopenia (ITP) (n = 40) and from controls (n = 35). Structural vWF was evaluated by multimer analysis. Children with D+ HUS had vWF:Ag of 2.53 and vWF:CBA of 1.98 U/mL. The corresponding values for patients with ITP were 1.35 and 1.82 U/mL, with CRI 1.55 and 1.55 U/mL, and with GE 1.68 and 2.10 U/mL; all values were higher than in controls (1.04 and 1.16 U/mL). The mean ratio of vWF:CBA to vWF:Ag ratio in controls was 1.13; only children with HUS had a dysfunctional vWF, as indicated by a low ratio of 0.78; the ratio was elevated in children with ITP (1.36) and GE (1.27) and was normal in those with CRI (1.06). No ultralarge molecular multimers of vWF were detected in any group, including HUS. The very high concentration of plasma vWF:Ag in HUS probably reflects endothelial cell damage or irritation. In contrast to all other groups, only children with HUS had a dysfunctional vWF, caused either by a primary (due to enterohemorrhagic Escherichia coli) or secondary (due to consumption of functionally active vWF) process. This abnormality was not obvious as structural anomaly by multimer analysis.     

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.     

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.     

Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII

In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.     

Pharmacokinetics of von Willebrand factor and factor VIII coagulant activity in patients with von Willebrand disease type 3 and type 2

Nine patients with von Willebrand disease type 3, six with type 2B, one with type 2A, and one patient with type 1/2N were infused with one dose of ≈50 or 100 IU ristocetin cofactor activity (RCoF) per kg body weight of von Willebrand factor (vWF) (Human), a product with a very low content of factor VIII (FVIII). Blood samples were collected over 96 h. The data for RCoF and vWF antigen (vWF:Ag) were fitted to a 1-compartment model decay. The data for FVIII:C were fitted to a model with a linear time 'synthesis' term and a 1-compartment decay. Results in von Willebrand disease type 3 patients (nine patients; 10 infusions) indicated a volume of distribution of 39.9 and 39.8 mL kg⁻¹ for RCoF and vWF:Ag, respectively. The FVIII:C rate of synthesis was 6.4 U dL⁻¹ h⁻¹ (range: 4.4–8.8). The decay rates for FVIII:C, RCoF, and vWF:Ag were 0.041 (h⁻¹) [ t _1/2: 16.9 h]; 0.061 (h⁻¹) [ t _1/2: 11.3 h] and 0.006 (h⁻¹) [ t _1/2: 12.4 h], respectively. In patients with von Willebrand disease type 2 ( n  = 8) the RCoF mean volume of distribution was 46 mL kg⁻¹. The factor VIIIC mean rate of synthesis was 5.5 U dL⁻¹h⁻¹ and the decay rate 0.043 (h⁻¹) [ t _1/2: 16.1 h]. The rate of decay for RCoF and vWF:Ag were 0.050 (h⁻¹) [ t _1/2: 13.9 h] and 0.044 (h⁻¹) [ t _1/2: 15.7 h], respectively.     

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.     

Direct radioimmune detection in human plasma of the association between factor VIII procoagulant protein and von Willebrand factor, and the interaction of von Willebrand factor-bound procoagulant VIII with platelets

The predominant procoagulant factor VIII (VIII:C) form in normal human plasma containing various combinations of anticoagulants and serine/cysteine protease inhibitors is a protein with mol wt 2.6 +/- 0.2 X 10(5). This protein can be detected by 125I-anti-VIII:C Fab binding and gel electrophoresis in the presence and absence of sodium dodecylsulfate (SDS) and is distinct from the subunit of factor VIII/von Willebrand factor (VIII:vWF) multimers. No larger VIII:C form is present in plasma from patients with severe congenital deficiencies of each of the coagulation factors, other than VIII:C. The mol wt approximately 2.6 X 10(5) VIII:C form is, therefore, likely to be the in vivo procoagulant form of VIII:C, rather than a partially proteolyzed, partially activated derivative of a larger precursor. About 60% of this procoagulant mol wt approximately 2.6 X 10(5) VIII:C form in plasma is present in noncovalent complexes with larger VIII:vWF multimers, which attach reversibly to platelet surfaces in the presence of ristocetin. This VIII:vWF-bound protein of mol wt approximately 2.6 X 10(5) may be the plasma procoagulant form of VIII:C which, after proteolytic activation, accelerates the IXa-mediated cleavage and activation of X postulated to occur on platelet surfaces.     

急性冠状动脉综合征血浆血管性血友病因子及其裂解酶

目的 观察急性冠状动脉综合征病人血浆血管性血友病因子（von Willebrand factor,vWF）及血管性血友病因子裂解酶（von Willebrand factor cleaving protease,vWF-cp）含量变化及临床意义.方法 急性冠状动脉综合征病人分不稳定型心绞痛组17例和急性心肌梗死组17例,分别于急性期（入院后即刻）和恢复期（4周）采血.稳定型心绞痛组和对照组各17例,入院次日空腹采血.应用酶联免疫吸附试验测定血浆vWF活性水平,残余胶原结合力试验测定血浆vWF-cp活性水平.结果 ①血浆vWF活性水平：不稳定型心绞痛组110%±25%、急性心肌梗死组急性期131%±25%,均高于稳定型心绞痛组84%±19%和对照组72%±13%（P＜0.01）;亦高于恢复期（不稳定型心绞痛组恢复期为77%±9%,急性心肌梗死组恢复期76%±11%）（P＜0.01）;②急性期血浆vWF-cp活性水平：不稳定型心绞痛组为53%±12%、急性心肌梗死组46%±12%,均低于稳定型心绞痛组77%±10%和对照组81%±17%（P＜0.01）;亦低于该组恢复期（不稳定型心绞痛与急性心肌梗死组恢复期分别为80%±7%和81%±7%,P＜0.01）;③恢复期各项指标与稳定型心绞痛病人和对照组相比差异无统计学意义（P＞0.05）.结论 血浆vWF水平升高、vWF-cp活性降低在急性冠状动脉综合征血栓形成中可能起重要作用.     

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.     

Increased von Willebrand factor antigen in the plasma of patients with vasculitis

The plasma concentrations of von Willebrand factor antigen (vWF:Ag) were determined in 101 patients who had the following diagnoses: vasculitis 8 patients, systemic lupus erythematosus (SLE) 51, rheumatoid arthritis (RA) 28, asthma 7, hereditary angioedema 7. The greatest mean concentration of vWF:Ag, 469% (normal 100% +/- 50), was observed in patients with vasculitis, often without elevation of the erythrocyte sedimentation rate. The mean concentration of vWF:Ag was also increased in both SLE (277%) and RA (194%). Twenty-four patients (15 with SLE, 6 with vasculitis, 3 with RA) had vWF:Ag concentrations greater than 300%. Four of these patients died within 1 year of the date of the study. Of the 15 SLE patients, 9 had vasculitis and 2 had active glomerulonephritis. The 3 RA patients had severe disease associated with extraarticular manifestations. Elevated vWF:Ag may reflect vascular damage, while markedly elevated levels of vWF:Ag appear to indicate a poor prognosis.     

Large experience with a factor VIII binding assay of plasma von Willebrand factor using commercial reagents

The present diagnostic assay for type 2N von Willebrand disease (VWD) is based on the in vitro measurement of the capacity of plasma von Willebrand factor (VWF) to bind exogeneous factor VIII (VWF:FVIIIB). We report a method using only commercially available reagents that is easy to perform. This method has been validated in a cohort of 144 patients with FVIII/VWF ratios < 0.6 using a plasma control mixture representative of intermediate VWF:FVIIIB. In total, 15 patients were diagnosed with markedly decreased VWF:FVIIIB and five patients were shown to have moderately decreased VWF:FVIIIB. Specific type 2N mutations were identified in all these patients.