Plasma Levels Of vWF Research Articles

Regulation of coagulation factors during liver regeneration in mice: Mechanism of factor VIII elevation in plasma

IntroductionsThe profiles of coagulation factor production during liver regeneration process remains to be fully elucidated. The present study was aimed to perform a comprehensive analysis whether hepatic gene expression was differentially regulated relative to the secretion of biologically active coagulation factors using a mouse model of liver regeneration. Materials and MethodsLiver regeneration was induced by performing a 2/3 partial hepatectomy (PHx). Plasma samples were assessed for coagulation factor activities (fibrinogen, prothrombin, V, VII, VIII, IX, X, XI, XII, and XIII) and the liver mRNA levels of coagulation, anti-coagulation, and fibrinolytic factors were quantified by real-time RT-PCR during the phase of liver regeneration. ResultsAt the peak of liver regeneration, the expression levels for all of the genes analyzed were found to be reduced in a time-dependent manner. Consistent with the gene expression levels, plasma activities of all coagulation factors, except for FVIII, were temporally declined during the same time frame. FVIII paradoxically demonstrated a significant increase (P<0.05) in plasma activities concomitant with the decrease of liver mRNA expression levels. We found that the increase in plasma FVIII activities might be associated with (1) a delay in the inactivation of plasma FVIII caused by increased VWF in plasma and decreased FVIII clearance in the liver, (2) the rapid release of FVIII from the storage sites, and (3) the alteration of intracellular trafficking pathway of FVIII. ConclusionsThe present study demonstrated that the process of liver regeneration involves a general reduction for many of the coagulation cascade proteins, but there are paradoxical increases in plasma levels of FVIII and VWF.

P.79 Preeclampsia: ABO blood groups and their relationship with FVIII, vWF and ADAMTS13 plasma levels

P.79 Preeclampsia: ABO blood groups and their relationship with FVIII, vWF and ADAMTS13 plasma levels

Expression patterns of plasma von Willebrand factor and serum interleukin-8 in patients with early-stage severe pulmonary contusion

von Willebrand factor (vWF) is only released from endothelial cells and platelets and is an in vivo and in vitro marker of endothelial injury in septic patients with acute lung injury (ALI). Interleukin-8 (IL-8), as a proinflammatory mediator causing recruitment of inflammatory cells, induces an increase in oxidant stress mediators and makes it as a key parameter for localized inflammation. However, it has not been well established whether the level of serum IL-8 is associated with the severity of lung injury and whether it is a prognosis marker for severe lung contusion. This study was to investigate the expression of plasma vWF and IL-8 and their association with the severity and outcomes of severe pulmonary contusion. A total of 63 patients were divided into a severe pulmonary contusion with acute respiratory distress syndrome (ARDS) group and a non-ARDS group, or a survivor group and a non-survivor group, or an injury severity score (ISS) <20 group and an ISS ≥20 group. Another 20 healthy volunteers served as controls. The levels of plasma vWF and serum IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) at 1, 3, 5 and 7 days after injury. The expression patterns of the plasma vWF and serum IL-8 were compared between different groups. The concentrations of plasma vWF and serum IL-8 were significantly increased in all severe pulmonary contusion patients at all time points in comparison with the control group. The concentrations of plasma vWF in patients with ARDS increased during the whole study period, but vWF in patients with non-ARDS increased gradually until day 5 and then decreased at day 7. The concentration of serum IL-8 showed a similar expression pattern in both groups, but the expression increased more significantly in the ARDS group than in the non-ARDS group. Interestingly, both plasma vWF and serum IL-8 levels steadily increased in the non-survivor group. Furthermore, the level of plasma vWF was higher in the ISS≥20 group than in the ISS<20 group. The level of serum IL-8 in the ISS≥20 group was consistently high, while that in the ISS<20 group peaked at day 3 and decreased at day 5. In addition, the level of plasma vWF was positively correlated with platelet count, but negatively correlated with oxygen index. The level of serum IL-8 was positively correlated with white blood cell count and ISS score, and inversely correlated with oxygen index. The elevated levels of plasma vWF and serum IL-8 in severe pulmonary contusion patients reflect the severity of pulmonary injury and patients outcomes, suggesting that the plasma vWF and serum IL-8 are sensitive markers for clinical evaluation of the severity of pulmonary injury and predication of patient prognosis.

Levels of von Willebrand factor and ADAMTS13 determine clinical outcome after cardioversion for atrial fibrillation

Von Willebrand factor (vWF) plays an essential role in platelet adhesion and thrombus formation. Patients with atrial fibrillation (AF) exhibit higher plasma vWF and lower ADAMTS13 antigen levels compared to controls. Little is known about vWF and ADAMTS13 in AF patients treated with cardioversion (CV). Thus we investigated the alterations of plasma vWF and ADAMTS13 after CV and evaluated the predictive value of these parameters for recurrence of AF. In this observational study we determined plasma levels of vWF and ADAMTS13 in 77 patients before and immediately after CV, as well as 24 hours (h) and six weeks thereafter, by means of commercially available assays. The vWF/ADAMTS13-ratio was significantly elevated immediately after CV (p=0.02) and 24 h after CV (p=0.002) as compared to baseline levels. ADAMTS13, 24 h after CV, exhibited a significant association with recurrence of AF (HR: 0.97; p=0.037). Accordingly, tertiles of ADAMTS13 showed a stepwise inverse correlation with the risk of recurrent AF (HR: 0.50; p=0.009). After adjustment for confounders, ADAMTS13 remained significant as an independent predictor of recurrent AF (HR: 0.61; p=0.047). Similarly, the vWF/ADAMTS13-ratio, 24 h after CV, was associated with rhythm stability and remained an independent predictor of recurrent AF (HR: 1.88; p=0.028). The regulation of vWF and its cleaving protease ADAMTS13 after CV might play a critical role in producing a pro-thrombotic milieu immediately after CV for AF. Since ADAMTS13 plasma concentration and the vWF/ADAMTS13-ratio are independently associated with rhythm stability, these indexes might be used for prediction of recurrence of AF.

ABO blood group as a potential risk factor for venous thromboembolism in acutely injured patients

Venous thromboembolism (VTE) is a major health problem that results in a significant burden on hospitals and patients. VTE screening and prophylaxis protocols in trauma patients vary significantly among hospitals and providers. In addition, many patients develop VTE even in the absence of "high-risk" categories. Therefore, more research is needed to better understand and prevent VTE in these patients. ABO blood group has long been recognised as a risk factor for VTE, but its contribution to VTE risk in the trauma setting is poorly studied. This paper reviews the literature describing the link between ABO blood group and VTE risk and the implications for VTE screening and prophylaxis in trauma patients. The effect of ABO blood groups are genotype-dependent - in most populations the A1 allele and the B allele increase risk while A2, O1, and O2 decrease risk of VTE. ABO group is a major determinant of plasma von Willebrand factor (vWF) and factor VIII levels, thereby (partially) mediating the effects of ABO blood group on VTE susceptibility. In addition, ABH antigens alter plasma levels of vWF via clearance mechanisms, which are in turn mediated by ADAMTS13. ABO blood group is a risk factor for VTE that warrants further investigation in trauma patients.

A Study of blood soluble P-selectin, fibrinogen, and von Willebrand factor levels in idiopathic and lone atrial fibrillation

A prothrombotic state with elevated levels of soluble P-selectin (sP-sel), fibrinogen, von Willebrand factor (vWf), and other haemostatic indices has been reported in some patients with atrial fibrillation (AF). Whether these changes are due to AF itself or coexistent cardiovascular diseases remains a matter of debate. Therefore, in the present study, the differences in plasma levels of sP-sel, fibrinogen, and vWf between patients with idiopathic/lone AF and sex-, age-, and risk factor-matched controls were investigated to determine whether AF itself might be associated with a hypercoagulable state. Ninety consecutive patients (63 males, 54.1 ± 10.1 years) with idiopathic AF were studied, 60 (43 males, 48.8 ± 7.5 years) of whom were diagnosed as lone AF. Plasma sP-sel and vWf were measured by enzyme-linked immunosorbent assay. Plasma fibrinogen was measured by chromometry. These indices in AF patients were compared with those in sex-, age- and risk factor-matched controls. Compared with the controls, patients with idiopathic AF had higher levels of sP-sel (AF vs. control: 33.4 ± 7.4 vs. 29.2 ± 6.5 ng/mL, P < 0.001) and fibrinogen (AF vs. control: 3.3 ± 0.9 vs. 3.0 ± 0.6 g/L, P = 0.02), but not vWf, whether with the adjustment of covariates or not. As for those < 60 years, between lone AF and age-matched controls, significant difference existed in the levels of sP-sel (AF vs. control: 34.5 ± 7.3 vs. 30.2 ± 7.3 ng/mL, P = 0.002), but not in those of fibrinogen and vWf, whether with the adjustment of covariates or not. Both platelet activation and abnormal changes in coagulation were suggested in idiopathic AF and a platelet activation state in lone AF. This supports the notion that AF per se contributes to a state of hypercoagulation.

Polymorphisms and Mutations in vWF and ADAMTS13 Genes and Their Correlation With Plasma Levels of FVIII and vWF in Patients With Deep Venous Thrombosis

Increased levels of factor VIII (FVIII) are a prevalent and independent risk factor for deep venous thrombosis (DVT) and are affected by von Willebrand factor (vWF) levels. ADAMTS13 contributes to vWF levels, and we investigated genetic polymorphisms previously described to be associated with decreased levels of these proteins in 435 patients with DVT (126 M and 309 F; median age 37 years, range 18-68 years) and 580 controls (163 M and 417 F; median age 35 years, range 18-68 years). Subsequently, we investigated the relationship between the genotypes and plasma levels of FVIII, vWF, and DVT risk. Patients with DVT showed higher plasma levels of FVIII:C, FVIII:Ag, and vWF:Ag (P < .001) when compared to controls. Patients and controls heterozygous for the 4751A>G polymorphism in the vWF gene presented decreased levels of vWF:Ag, FVIII:Ag, and FVIII:C (P < .001), but this was not a protective factor for DVT. Individuals heterozygous for 1852C>G polymorphism in ADAMTS13 gene, which is associated with reduced levels of ADAMTS13, had significantly elevated levels of vWF:Ag (P = .001), FVIII:Ag (P = .01), and FVIII:C (P = .02). However, this polymorphism was not a risk factor for DVT in our study. Heterozygosis for a new polymorphism identified in ADAMTS13 gene, 1787-26G>A, was significantly associated with elevated levels of FVIII:C (P = .02) when compared to wild type. Despite the tempting assumption that genetic factors that change ADAMTS13 activity might modulate the risk of DVT by altering vWF and FVIII levels, the polymorphisms analyzed in this study did not correlate with DVT risk among patients investigated.

Prevalence of Parental Thrombophilic Defects After Fetal Death and Relation to Cause

To estimate whether parental thrombophilic defects after fetal death, either acquired or inherited, were more prevalent than in the normal population and to estimate associations between these thrombophilic defects and different fetal death causes. In a multicenter, prospective cohort study of 750 fetal deaths, we tested couples for antithrombin, protein C, total and free protein S, and von Willebrand factor (vWF) plasma levels. Mothers' values were compared with reference values in gestational age-matched healthy pregnant women, and fathers were compared with healthy men. Prevalence of factor V Leiden, prothrombin G20210A mutation, and lupus anticoagulant were compared with the normal population. A panel classified death cause. More women with fetal death had decreased antithrombin (16.8%, P<.001) and protein C (4.0%, P=.03) and increased vWF (15.5%, P<.001) plasma levels than healthy pregnant women (2.5%). However, compared with normal ranges in the nonpregnant population, we only observed more women with increased vWF (12.4%, P<.001). More fathers had decreased free protein S (6.3%, P<.001) and elevated vWF (12.1%, P<.001) than healthy men (2.5%). Prevalence of inherited thrombophilias was not higher in couples with fetal death than in the population. Neither inherited nor acquired maternal or paternal thrombophilic defects were associated with the main cause of death. Of placental causes, abruption and infarction were associated with acquired maternal defects. Except for vWF and paternal free protein S, acquired and inherited thrombophilic defects were not more prevalent after fetal death. Routine thrombophilia testing after fetal death is not advised. II.

The impact of acute atrial fibrillation on the prothrombotic state in patients with essential hypertension

Objectives To investigate whether acute atrial fibrillation (AF) creates a prothrombotic state in hypertensive patients, and to assess the evolution in research indices after cardioversion. Design and methods Plasma levels of von Willebrand factor (vWf), soluble P-selectin and fibrin D-dimers were measured in hypertensive patients with acute AF pre-cardioversion and at 1, 7, 14, and 30 days post-cardioversion. The results were compared with hypertensive controls and healthy controls. Results Pre-cardioversion plasma vWf levels in acute AF patients were similar to those of controls; however, post-cardioversion, the vWf levels increased significantly and only returned to baseline levels 14 days later. Compared with controls, acute AF patients had higher levels of fibrin D-dimers, which lasted at least 14 days after cardioversion. Conclusions This study demonstrated that hypertensive patients with acute AF have an abnormal prothrombotic state, which persists for up to 14 days after cardioversion.

Association of ABO(H) and I blood group system development with von Willebrand factor and Factor VIII plasma levels in children and adolescents

The modulation of Factor (F)VIII activity (FVIII : C), von Willebrand factor antigen (VWF : Ag), and von Willebrand factor ristocetin cofactor (VWF : RCo) by the ABO(H) blood group is well established in adults. Expression of ABH antigens on N-linked glycans of VWF protects plasma VWF from proteolysis and clearance. Protection by H antigens is less effective than by AB antigens, resulting in approximately 25% lower VWF plasma levels in adults with blood group O compared to non-O. Given the reduced branching of ABO(H) bearing structures (I blood group system) with lower numbers of H, A, and B antigen sites during the first 18 months of life, we reasoned that if the relationship between ABO(H) blood group and VWF levels were causal, the difference of ABO(H) blood group-dependent VWF levels should be marginal or not be observed in the first months of life. We undertook quantification of FVIII : C and VWF in 574 presumably healthy children aged 1 to 210 months and correlated the values with ABO(H) blood type. Moreover, we establish reference intervals for common coagulation variables for several pediatric age groups. Significant differences between blood group O versus non-O values of FVIII : C, VWF : Ag, and VWF : RCo were not observed in the first months of life, started to develop during childhood, and in adolescence reached adult values. In comparison to the levels for adults and adolescents, we report fundamental differences of VWF levels in the first year of life, which may be associated with the physiologic development of the ABO(H) and I blood group system.

Leukocyte telomere length is inversely correlated with plasma Von Willebrand factor

Introduction Leukocyte telomere length (LTL) is short, while the plasma level of Von Willebrand (VWF) is high in persons with atherosclerosis. Moreover, both short LTLs and high VWF levels are observed in individuals who display risks for atherosclerosis, including hypertension, obesity, insulin resistance, cigarette smoking and low socio-economic status. We examined the association between LTL and VWF plasma levels to test the hypothesis that high levels of VWF promote an increase in the turnover of blood cells, including leukocytes. Such a process would heighten the rate of age-dependent LTL attrition, ultimately resulting in shortened LTL. Methods We studied 3 cohorts: the ADELAHYDE study (age 60–87 years), the ERA study (age 41–88 years) and the Longitudinal Study of Aging Danish Twins (LSADT) (age 73–94 years). Results Multiple regression analysis with LTL as the dependent variable, and age, sex and VWF as the independent variables showed that LTL was inversely correlated with VWF in the ADELAHYDE (beta = − 0.125, p < 0.001) and the ERA study (beta = − 0.148, p = 0.010). The LSADT displayed VWF x age interaction, which was incorporated into the model, showing that LTL was also inversely correlated with VWF (beta = − 0.057, p = 0.04). Conclusions The inverse relationship between LTL and VWF, observed in 3 different populations, suggests that LTL might be linked to the coagulative status of the individual. Further research will be required to confirm our observations and their clinical ramifications.

Correlation between plasma asymmetric dimethylarginine and different types of coronary heart disease.

To monitor the changes of plasma asymmetric dimethylarginine (ADMA), nitric oxide (NO), and von Willebrand factor (vWF) levels in patients with stable angina pectoris (SAP) or acute coronary syndrome (ACS) and to evaluate the correlation between ADMA and different types of coronary heart disease. A total of 143 subjects were divided into a non-CHD group, a SAP group and an ACS group. Plasma levels of ADMA, NO and vWF were examined and their correlation with SAP or ACS was analyzed. Compared with the non-CHO or the SAP group, ADMA level was elevated in the ACS group (P<0.05). The ADMA level tended to increase in the SAP group compared with the non-CHD group, but had no significant difference (P>0.05). Compared with the non-CHD group, NO level was decreased in both the SAP and ACS group (P<0.05), and it decreased more in ACS group than that in the SAP group (P<0.05); vWF levels were increased in both the SAP and ACS group compared with the non-CHD group (P<0.05). There was no significant difference in the plasma levels of vWF in the SAP and the ACS group (P>0.05). The change of plasma ADMA level is closely correlated with acute coronary syndrome. ADMA might be a clinical marker for acute coronary syndrome.

Clinical Summaries

Clinical Summaries

Establishment of an assay for active von Willebrand factor in plasma and its application

Objective To establish an double-antibody sandwich method to detect the amount of active vWF in the plasma of healthy people and diabetic patients.Methods A double-antibody sandwich method has been established using llama-derived anti-active vWF antibody and horseradish peroxidaselabelled rabbit anti-human vWF polyclonal antibodies.The study randomly selected 66 healthy people,30 diabetic patients,10 cases of von Willebrand disease 2B(vWD-2B),5 cases of congenital thrombotic thrombocytopenic purpura(TIP),13 cases of acquired TIPs and 13 cases of malaria.The activated vWF level were described as the ratio of absorbance value(A)representing activated vWF value using AU/vWFa-11 antibody-based ELISA method to the concentrations of vWF antigen.At the same time,the absorbance values representing the concentrations of active vWF were measured when the concentrations of vWF antigen were 62,125,250μg/L respectively.The data were analyzed with GraphPad Prism 4 software.Under the premise of reaching a good linear regression,plasma active vWF values were calculated as the slope ratio of the sample plasma to the standard plasma.Accordingly to the ratio evaluated the levels of active vWF in plasma.Results The coefficients of variation(CVs)for intra assays were 4.21%,4.98% and 6.32% respectively.The CVs for interassays were 6.34%,7.02% and 7.69% respectively.In healthy adults,the level of active vWF in plasma was 0.74±0.35.There was no significant difference of the vWF levels[male (7.29±5.15)×10~3μg/L,female(9.42±4.67)×10~3μg/L]and active vWF levels(male 0.74±0.34,female 0.73±0.35)between male and female in healthy people(t=1.35,0.04,P＞0.05).The vWF levels of the group over 40 years old[(10.64±5.39)×10~3μg/L]were higher than these below 40 years old[(6.11 ±2.84)×10~3 μL)(t=8.24,P ＜ 0.01)],but there was no significant difference of the active vWF levels difference between these groups(over 40 years old 0.70±0.38,under 40 years old 0.78 ± 0.29,t=1.02,P＞0.05).The plasma levels of active vWF in vWD-2B type,congenital TIP,acquired TIP,malaria and diabetic groups(1.07±0.66,6.52±1.30,5.82±1.47,1.53±0.11,2.17±0.53)were higher than those in the control group(F=10.23,P ＜ 0.01).Conclusions Double-antibody sandwich method for detection of active vWF levels in plasma had good reproducibihty and specificity,which could be used to indicate the true active vWF levels under disease state.It could also provide an important contribution to analyze the mechanism for the active vWF in plasma. Key words: von Willebrand factor; Enzyme-linkedimmunesorbentassay; Purpura,thrombocytopenic

Plasma levels of Von Willebrand factor are increased in patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterised by inappropriate hypertrophy, small-vessel coronary artery disease, myocyte disarray and increased interstitial fibrosis. Microvascular dysfunction is a common finding in HCM and its extent has been proposed as an important prognostic marker. Plasma von Willebrand factor (vWf) is an established marker of endothelial damage or dysfunction; however it has scarcely been studied in HCM. We hypothesised that vWf could be raised in patients with HCM and be related to different variables associated with severity of HCM. Methods We included 124 HCM patients, 93 males, aged 48 ± 15 years, 59 healthy control subjects with similar age and sex and 20 patients with ischemic heart disease but clinical stability for the last 6 months. A complete history and clinical examination was performed, including 12-lead electrocardiogram, echocardiography, 24 hours ECG-Holter monitoring, and symptom limited treadmill exercise test. Risk factors for sudden death were evaluated. A blinded cardiac MRI was performed with late enhanced study with Gadolinium. Plasma vWf levels were assayed by commercial ELISA. Results Patients showed higher levels of vWf (140.0 ± 65.0 UI/ml vs 105.0 ± 51.0 UI/ml, p < 0.001) even after adjusting for ABO blood group. vWf levels were found raised in patients with severe functional class (168.4 ± 65.9 UI/mL vs 132.4 ± 60.7 UI/mL, p = 0.020), atrial fibrillation (175.8 ± 69.4 UI/mL vs 133.0 ± 59.0 UI/mL, p = 0.005), hypertension (161.4 ± 60.8 vs 128.9 ± 60.5, p = 0.010) obstruction (153.9 ± 67.9 vs 128.2 ± 57.4 UI/mL, p = 0.046) and non sustained ventricular tachycardia (159.3 ± 59.1 vs 133.0 ± 63.0, p = 0.049). vWf correlated with age (r:0.26; p = 0.006) and obstruction (r:0.22; p = 0.021). Conclusions We show, for the first time, patients with HCM present significantly raised levels of vWf. These are associated with different conditions related to the severity of the disease.

Alterations in biomarkers of endothelial function following on‐pump coronary artery revascularization

Cardiopulmonary bypass (CPB) has been associated with activation and injury of endothelial cells, probably responsible for the systemic inflammatory response syndrome (SIRS) taking place in these patients. We measured plasma concentrations of soluble P-selectin (sP-s), E-selectin (sE-s), tetranectin (TN), vonWillebrand factor (vWF) levels, and angiotensin-converting enzyme (ACE) activity in 31 adult patients undergoing elective coronary artery bypass grafting, just before and up to three days after surgery, and in 25 healthy volunteers. Patients showed higher plasma sP-s and sE-s and ACE concentrations, just before surgery, but significantly lower TN levels, compared with controls. During the first three postoperative days (PD), the concentration of each of the molecules followed a different and independent pattern, although in the third PD, the levels of sP-s, sE-s and ACE were higher and those of vWF and TN lower, compared with the preoperative ones. However, patients had higher sP-s (P=0.06), sE-s (P=0.07), and vWF (P=0.005), but lower TN concentrations (P=0.02) on the third PD compared with controls. CPB is characterised by pronounced changes in plasma sP-s, sE-s, TN, vWF levels, and ACE activity, which are associated with significant alteration in the intra- and early postoperative endothelial function observed in open heart surgery.

The Role of the VWF A1 and A2 Domains in Murine Models of TTP and Thrombosis.

Abstract 3060Poster Board II-1036von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein synthesized in endothelial cells and megakaryocytes. In humans and mice, VWF dysfunction is associated both with defects in hemostasis, and with the systemic blood clotting disease thrombotic thrombocytopenic purpura (TTP). The initial adhesion of platelets to sites of vascular injury in large part involves binding of the VWF A1 domain to the platelet glycoprotein receptor GPIb alpha. This VWF A1-GPIb alpha interaction, along with deficiency of the ADAMTS13 plasma metalloprotease, is thought to be required for the pathogenesis of TTP. Deficiency of ADAMTS13 results in the failure to cleave the Y1605-M1606 sissile bond within the A2 domain of VWF. The structure of VWF is strongly influenced by its high content of cysteine residues, all of which are involved in inter-or intra-chain disulfide bonds. The location of these cysteine residues within the A domains leads to the formation of disulfide loops within the A1 (Cys 1272-1458) and A3 (Cys 1686-1872) domains, but not within the A2 domain. The lack of a disulfide loop allows the A2 domain to assume a “flexible” conformation that is thought to “open” in response to fluid shear stress, exposing the Y1605-M1606 bond to cleavage by ADAMTS13. To investigate specifically the role of the VWF A1-Gp1b alpha interaction in the context of otherwise functional VWF in vivo, we generated a chimeric murine VWF expression construct in which the murine A1 domain sequence is replaced with the corresponding sequence from human VWF (the human VWF A1 domain is known to not interact appreciably with murine GPIb alpha). Additionally, we engineered a VWF construct in which paired cysteine residues analogous to those in the A1 and A3 domains were introduced into the A2 domain sequence, with the goal being to “lock” the A2 domain closed and prevent cleavage by ADAMTS13. Hydrodynamic tail vein injection of both the VWF-hA1 and the VWF-A2 lock constructs into VWF-deficient mice resulted in plasma VWF levels up to 20-fold higher than observed in wild-type mice, dependent on the amount of plasmid injected. Importantly, the degree of VWF multimerization appeared nearly identical both to that observed in wild-type mice, and to mice injected with wild-type murine VWF, and expression persisted for approximately 30 days. Functionally, unlike WT murine VWF, expression of VWF-hA1 failed to restore thrombus formation in a ferric chloride-induced injury model, demonstrating the crucial importance of the VWF A1-GP1b apha interaction in thrombus formation. Currently we are investigating whether expression of VWF-hA1 can support disease pathogenesis in a mouse model of TTP. Similarly, we are determining whether expression of VWF-A2 lock leads to development of TTP, even in the presence of ADAMTS13. The ultimate goal of these studies is to completely “humanize” the VWF A1-GP1b alpha interaction in mice by replacing the murine GP1b alpha sequence with that from humans. These resulting animals will be used to further investigate the role of the VWF A1 domain-GPIb alpha interaction in vivo, and should prove useful for identifying compounds to effectively inhibit this interaction in humans. In addition, the expression of a VWF construct that is unable to be cleaved by ADAMTS13 should help to elucidate the role of VWF cleavage in TTP pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

Compromised Proteolytic Cleavage of Von Willebrand Factor Type 2N Variants by ADAMTS13 in the Presence of Factor VIII (and Platelets) Under Fluid Shear Stress.

Abstract 28von Willebrand Factor (VWF) is a large, multimeric adhesive glycoprotein that is involved in the formation of a platelet plug after vascular injury. In addition, VWF functions as a carrier protein for clotting factor VIII (FVIII) which prevents rapid clearance of plasma FVIII. Decreased levels of VWF or defects in VWF function are found in patients with von Willebrand disease (VWD). Quantitative defects of VWF protein in type 1 and 3 VWD affect plasma levels of both VWF and FVIII, whereas qualitative defects in type 2 VWD result in abnormal binding of VWF to platelets such as in type 2A, 2B, and 2M, or to FVIII such as in type 2N. Previous studies have demonstrated that FVIII binds VWF, which dramatically accelerates the proteolytic cleavage of multimeric VWF by ADAMTS13 under mechanically-induced shear stresses. This rate-enhancing effect of FVIII on VWF proteolysis appears to depend on the ability of FVIII to bind VWF, as a FVIII variant lacking the A3 acidic region fails to exhibit the cofactor activity that accelerates VWF proteolysis. To determine whether reduced FVIII binding in VWF type 2N variants affects VWF proteolysis, we examined the proteolytic cleavage of recombinant VWF type 2N variants in the presence of FVIII (and lyophilized platelets) for variants with a moderate VWD phenotype (Arg854Gln and His817Gln) or severe VWD phenotype (Arg763Gly, Arg782Trp, Thr791Met, and Arg782Trp + His817Gln). Recombinant VWF type 2N variants (37.5 μg/ml or 150 nM) were incubated with ADAMTS13 (25 nM) in the absence and the presence of various concentrations of FVIII (0-40 nM) with or without lyophilized platelets (0-600×103/μl) under fluid shear stress. The proteolytic cleavage products (350 kDa) were determined by 5% SDS-PAGE and Western blot under denaturing but non-reducing conditions. We show that the proteolytic cleavage of VWF type 2N variants by ADAMTS13 under these conditions was variably reduced as compared that of wild type VWF. The reduction in the cleavage rate was proportional to the degree of reduction in VWF FVIII binding activity, which was assessed with a microtiter assay, with the least cleavage by ADAMTS13 of the variants with the lowest FVIII binding activity. This reduced cleavage of the type 2N variants was not correlated with the binding affinity between the type 2N variants and ADAMTS13 protease. These results provide further evidence that binding of FVIII to VWF, which may alter VWF conformation, is necessary to accelerate VWF proteolysis by ADAMTS13 under fluid shear stress. This variability in ADAMTS13 cleavage may contribute to the heterogeneity of bleeding phenotype of type 2N VWD variants. The bleeding phenotype may be modulated not only by plasma FVIII levels, but also the extent of VWF proteolysis by ADAMTS13 under physiological fluid shear stress. Disclosures:No relevant conflicts of interest to declare.

Presence of a Common Haplotype in the Turkish VWD Patients.

Abstract 4435Von Willebrand Factor (VWF) is the cruical molecule in the initiation of hemostatic response to vascular injury. Quantitative or qualitative defects in VWF are associated with Type 1/Type 3 or Type 2 von Willebrand disease (VWD), respectively. VWF is encoded by the VWF gene composed of 52 exons and located on chromosome 12. Nonfunctional copy of the VWF gene containing a region from exon 22 to exon 34 is located on chromosome 22. Plasma VWF levels show a five-fold range within a normal population and genetic varaiablity at the VWF locus accounts for 30% of this variance. VWF gene exhibits a high variablity in its sequence. More than 150 polymorphisms and more than 200 different mutations have been reported in VWF database. Most of the polymorphisms and mutations are detecetd in the region that is duplicated in the pseudogene. Genetic recombination between the gene and its pseudogene may account for this high variability in the VWF gene. Highly polymorphic structure of the VWF gene suggests that haplotypic interactions may account for the variable expressivity of the gene. In this study we report presence of a common haplotype structure in exon 28 of the VWF gene in the Turkish VWD patients. Patient population includes Type1, Type 3 and Type 2 VWD patients. DNA sequencing analysis of 32 VWD patients revealed presence of a novel c.4453G>C change in exon 28 of the 23 patients. c.4453G>C change results in the p.1485V>L substitution. Individuals having p.1485V>L substitution was also carrier for the previously identified c.4500G>A (p.1500A), 4503C>T (p.1501F), c.4508T>C (p.1503L>P), and c. 4517C>T (p.1506S>L) changes in exon 28. c.4500G>A, 4503C>T changes were reported as polymorphisms and c.4508T>C and c. 4517C>T changes were reported as mutations identified in VWD Type 2A patients in VWF database. To determine whether these alterations represent a haplotype, we are currently analyzing exon 28 of the parents of the carrier patients. In 8 of the 11 patient families analyzed, patients inherited the haplotype from the parents. These preliminary data suggests presence of a common haplotype for the Turkish VWD patients. Future studies with the rest of the patient families and normal population will determine whether this haplotype is a benign haplotype or associated with VWD. Disclosures:No relevant conflicts of interest to declare.

Similar immune profile in bipolar disorder and schizophrenia: selective increase in soluble tumor necrosis factor receptor I and von Willebrand factor

Alterations in the inflammatory system have been associated with schizophrenia and major depression, while bipolar disorder has been less studied. Most previous studies examined small samples, and the literature is inconsistent with regard to specific underlying immune mechanisms. In the present study, we examined markers representing different inflammatory pathways, and the aim was to investigate whether the levels of inflammatory parameters in a representative sample of bipolar disorder and schizophrenia are elevated compared to healthy controls, and to investigate whether the inflammatory profile is different between the groups. Plasma levels of soluble tumor necrosis factor receptor 1 (sTNF-R1), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), high-sensitivity CRP (hs-CRP), soluble CD40L ligand (sCD40L), and von Willebrand factor (vWf) were measured with ELISA techniques in a catchment area based sample of consecutively referred patients with severe mental disorders [N = 311, comprising bipolar disorder (n = 125) and schizophrenia (n = 186)] and in healthy volunteers (n = 244). Plasma levels of sTNF-R1 and vWf were statistically significantly increased in both bipolar disorder and schizophrenia compared to controls (p < 0.00001), and were also increased in unmedicated patients, but there were no major differences between the two diagnostic groups. Controlling for age, gender, ethnicity, cardiovascular disorders, kidney and liver function, and other confounders did not affect the results. There were no differences in other inflammation factors between the groups. The present results indicate specific alterations of endothelium-related inflammation processes in both bipolar disorder and schizophrenia.