Von Willebrand Factor Propeptide Research Articles

Markers of endothelial dysfunction differ between subphenotypes in children with sickle cell disease

In adult patients with sickle cell disease two distinct subphenotypes have previously been defined: patients with the viscosity-vaso-occlusion subphenotype (VVO) suffer mainly from vaso-occlusive pain crises and have a relatively high hemoglobin concentration. Patients classified as the hemolysis-endothelial dysfunction subphenotype (HED) suffer from stroke and pulmonary hypertension and have an elevated concentration of lactate dehydrogenase. However, this classification is not possible in children due to low rates of complications. We used laboratory markers to classify children into the two subphenotypes, and measured vWF and vWF propeptide as markers of endothelial dysfunction. We included 106 children with sickle cell disease (mean age 8.7years), 74 (70%) with HbSS/HbSβ° genotype and 32 (30%) with HbSC/HbSβ(+) genotype. vWF and vWF propeptide were significantly elevated in patients with sickle cell disease; this was more pronounced in patients with the HbSS/HbSβ° genotype. Patients with the HED subphenotype had higher levels of vWF propeptide, and a trend towards higher levels of vWF compared to those with the VVO subphenotype. We demonstrated that even young children in a stable clinical condition show signs of persistent endothelial dysfunction. A prospective study should demonstrate whether elevated levels of vWF and its propeptide are associated with an increased risk of complications specific for the HED subphenotype.

Quantitative and qualitative changes of von Willebrand factor and their impact on mortality in patients with end-stage kidney disease

von Willebrand factor (vWF) antigen levels are elevated in patients with end-stage kidney disease (ESKD). We determined the quantitative and qualitative abnormalities of vWF and factors influencing vWF proteolysis in participants with ESKD compared with age-matched controls and determined the association between abnormalities in vWF and mortality over 4 years of follow-up. vWF : Ag and von Willebrand factor propeptide (vWFpp) levels, vWF functional activity (vWF :RCo), vWF multimer profiles, ADAMTS-13, thrombospondin 1 (TSP-1), and interleukin 6 (IL-6) were evaluated before and after a single hemodialysis treatment in 55 individuals with vascular disease and an age-matched group of controls (n = 21). vWF : Ag and vWF activity were significantly higher in the ESKD patients and levels increased further following the dialysis procedure. The percentage of high molecular weight multimers (%HMWMs) was significantly elevated in the ESKD patients compared with controls. TSP-1 was lower and IL-6 was higher providing possible explanation for the increase in %HMWM in ESKD. The %HMWM dropped significantly in the postdialysis sample. Mortality at 4 years was significantly associated with vWF : Ag. There are higher plasma vWF : Ag levels and a small increase in HMWMs in the ESKD milieu. The acute drop in the %HMWM of vWF postdialysis appears to be due to shear forces encountered during the dialysis procedure. The contribution of these abnormalities to either a pro-thrombotic and/or pro-bleeding phenotype in this population requires further study.

Coagulopathy in malaria

Blood coagulation activation is frequently found in patients with malaria. Clinically apparent bleeding or disseminated intravascular coagulation (DIC) is associated with very severe disease and a high mortality. Protein C, protein S, and antithrombin levels were found to be low in P. falciparum, but were normal in P. vivax infection. Plasma levels of plasminogen activator inhibitor-1 were high in cases of P. falciparum infection whereas tissue plasminogen activator levels were low. Elevated plasma levels of von Willebrand factor (vWF) and vWF propeptide, thrombomodulin, endothelial microparticles have been reported in P. falciparum-infected patients. It has been demonstrated that severe P. falciparum infection is associated with acute endothelial cell (EC) activation, abnormal circulating ultralarge vWF multimers, and a significant reduction in plasma ADAMTS13 function. These changes may result in intravascular platelet aggregation, thrombocytopenia, and microvascular disease. It has also been shown that P. falciparum-parasitized red blood cells (pRBCs) induce tissue factor (TF) expression in microvascular ECs in vitro. Recently, loss of endothelial protein C receptor (EPCR) localized to sites of cytoadherent pRBCs in cerebral malaria has been demonstrated. Severe malaria is associated with parasite binding to EPCR. The cornerstone of the treatment of coagulopathy in malaria is the use of effective anti-malarial agents. DIC with spontaneous systemic bleeding should be treated with screened blood products. Study in Thailand has shown that for patients who presented with parasitemia >30% and severe systemic complications such as acute renal failure and ARDS, survival was superior in the group who received exchange transfusion. The use of heparin is generally restricted to patients with DIC and extensive deposition of fibrin, as occurs with purpura fulminans or acral ischemia. Antiplatelet agents interfere with the protective effect of platelets against malaria and should be avoided.

EComment. Potential therapeutic agents in vasoplegic syndrome after cardiac surgery

Vasoplegic syndrome (VS) or vasodilatory shock frequently occurs after cardiac surgery, particularly with cardiopulmonary bypass(CPB). It is often associated with high morbidity and mortality, especially if it is resistant to intravascular volume expansion and conventional vasoconstrictive drugs [1]. This complication is similar to septic shock and characterized by severe and persistent hypotension, decreased systemic vascular resistance (SVR), normal or increased cardiac output (CO), low intra-cardiac filling pressures, and the necessity for high-dose catecholamines. Until today, the aetiology of VS is not fully understood. However, this syndrome is thought to be caused by the inflammatory-mediated dysregulation of endothelium homeostasis and subsequent endothelial dysfunction, including endothelial cell activation, endothelial permeability augmentation, coagulation defects, production of different regulators, and vascular tone disorders [1]. We read with great interest the paper by Kortekaas et al [2]. The authors report that pre-existing endothelial cell activation, reflected by higher baseline von Willebrand factor pro-peptide and soluble P-selectin levels is a predisposing factor for VS after mitral valve surgery. They also conclude that this activation may have resulted in desensibilization of endothelium in patients with VS. We would like to add some comments on possible agents for the management of VS VS is attributed to a combination of endothelial injury, arginine-vasopressin system dysfunction, and the release of vasodilatory inflammatory mediators[1]. Although these markers may be useful for the identification of patients at increased risk of developing VS, effective treatment strategies for this syndrome are currently lacking. The choice of vasoconstrictive agents for VS is a matter of controversy. To maintain adequate haemodynamics for VS, the conventional treatment based on fluid resuscitation and catecholamine drugs is ineffective in a subset of patients with profound vasodilatation[1]. Arginine vasopressin (AVP) and methylene blue (MB) may be used as additional rescue or a preventative treatment for VS [1, 3]. AVP is well-known to treat norepinephrine-resistant vasodilatation but may be associated rebound hypotension after discontinuation [3,4]. AVP significantly increases mean arterial pressure (MAP) and SVR, and results in a marked decrease in norepinephrine doses [1]. Tayama and associates [3] treated postoperative refractory low SVR hypotension concomitant with pulmonary hypertension by AVP. They reported that exogenous AVP normalized SVR and increased MAP. They also observed that AVP enhanced urine output and improved respiratory function. Noto and co-workers [4] reported that they treated six patients with terlipressin (TP; 1 mg bolus), a synthetic analogue of AVP for post-CPB refractory vasodilatory hypotension. In these patients, the authors suggested that exogenous TP administration normalized SVR and increased MAP with a minimum effect on pulmonary pressure. MB, an inhibitor of nitric oxide synthase and guanylate cyclase, seems to be a possible effective therapeutical option and to improve outcomes in patients with catecholamine-resistant VS after cardiac surgery [1, 5]. In a prospective observational study, Leyh and colleagues [5] tested the effect of MB (2 mg/kg over 20 minutes) in 54 patients with norepinephrine-resistant VS. In this study, MAP increased and norepinephrine dose decreased 1 hour after MB administration. These changes were accompanied by a significant decrease in CO. Finally, we think that further studies are necessary to find the most appropriate possible therapeutic agents for VS. Conflict of interest: none declared

P.P2063S: a neutral VWF variant masquerading as a mutation

Dear Editor, Kasatkar and colleagues recently reported that the previously described von Willebrand factor (VWF) variant c.6187C>T (p.P2063S) appears to be a common cause of type 3 von Willebrand disease (VWD3) in the Indian population [1]. In support of their conclusion, p.P2063S was the only VWF variant observed in 11 Indian VWD3 index cases (IC), tracked with disease phenotype and was predicted to be deleterious via in silico protein prediction analysis. In order to ensure accurate molecular characterisation of VWF variants, it is essential to consider all available information. Regarding p.P2063S, there are several lines of evidence that contradict the conclusion drawn. p.P2063S is also observed in healthy controls with reported population frequencies between 0.6–2.5% in African-Americans / Caucasians [2–4] and (as reported by Kasatkar and colleagues [1]) 3% in Indians. Given a frequency of 3% in the general population, Hardy-Weinberg equilibrium predicts homozygosity for this variant in approximately 1/1100 individuals. This would suggest a high population frequency of VWD3 if this variant were causative. It is also notable that p.P2063S has not been reported as causal in previous studies of VWD3 in Indians [5–7]. The majority of missense mutations reported in VWD3 patients occur in the VWF propeptide and C-terminus where they are reported to affect protein folding and multimerisation resulting in intracellular retention of the protein [2,7,8]. Kasatkar and colleagues argue that diagnosis of VWD3 relies on defective release or synthesis of VWF [1], however given that p.P2063S has no effect on VWF expression in vitro [2] it seems unlikely that this variant would affect VWF in vivo. This is supported by observations of p.P2063S in VWD patients inherited together with other VWF variants that more readily explain their VWD phenotype (Table 1), most notably in a Turkish VWD3 IC [8]. Table 1 Co-inheritance of p.P2063S and actual VWD causing variants In previous Indian VWD3 studies [5–7], molecular analysis failed to fully account for the genetic cause in 8/54 IC (15%). It is therefore likely that the actual disease causing mutation has yet to be identified by Kasatkar and colleagues, who imply that DNA scanning methods were used to identify mutations in their patient cohort [1]. DNA scanning methods are not 100% sensitive to all sequence variations so may fail to detect the ‘true’ mutation, for example chemical cleavage mismatch analysis identified a homozygous p.P2063S variant in a Turkish VWD3 IC but failed to identify the actual p.Q1734* causative mutation (Table 1) [8]. Alternatively, the causative mutation may have been overlooked if, for example, it were a synonymous variant that disrupts normal splicing or protein folding, or a deep intronic variant causing disruption of splicing outside the VWF regions analysed [9]. In summary, review of the available information supports the conclusion that p.P2063S is a common neutral VWF polymorphic variant. Further studies (for example using mRNA as the template investigated) may identify the underlying cause of VWD in this interesting patient group.

Pre-existing endothelial cell activation predicts vasoplegia after mitral valve surgery†

Post-cardiac surgery vasoplegia is a common complication of cardiac surgery, characterized by profound loss of systemic vascular resistance. This results in severe hypotension, high cardiac output and metabolic acidosis reflecting inadequate tissue perfusion. The pathophysiological mechanisms underlying this syndrome remain unknown. We hypothesized that this vasoplegia reflects endothelial dysfunction, either as pre-existing condition or as a consequence of the surgical procedure. To examine these mechanisms, six established and distinct markers of endothelial cell activation were measured pre- and perioperatively in patients undergoing mitral valve surgery. Arterial (radial artery) and myocardial venous blood samples (coronary sinus) were collected simultaneously over the reperfused heart at various time points during the first hour after reperfusion. Additional samples were collected at baseline (brachial vein) and 1 day post-reperfusion (radial artery). Post-cardiac surgery vasoplegia was defined as a mean arterial blood pressure of <60 mmHg, with a cardiac index of ≥2.2 l/min/m(2) treated with continuous intravenous administration of norepinephrine. No myocardial release of endothelial cell activation markers was observed upon reperfusion in patients with vasoplegia (n = 15; mean age 71 years, 73% male). In contrast, in patients without vasoplegia (n = 24; mean age 64 years, 54% male), reperfusion was characterized by a myocardial release of three endothelial cell activation markers. Myocardial von Willebrand Factor propeptide, osteoprotegerin and interleukin-8 were increased 107% (P < 0.001), 106% (P = 0.02) and 116% (P = 0.009), respectively, compared with arterial levels upon reperfusion. Similar systemic levels of all markers were found upon reperfusion in both groups, except for 120% increased soluble P-selectin (sP-selectin) levels in vasoplegia patients (P = 0.03). Remarkably, postoperative vasoplegia was identified with baseline von Willebrand Factor propeptide levels with a cut-off value of 11.9 nM as well as with baseline sP-selectin levels with a cut-off value of 64.4 ng/ml. Pre-existing endothelial cell activation, reflected by higher baseline von Willebrand Factor propeptide and sP-selectin levels, is a predisposing factor for post-cardiac surgery vasoplegia. The pre-existing endothelial cell activation may have resulted in desensibilization of endothelium in patients who develop vasoplegic syndrome, resulting in no myocardial release of endothelial cell activation markers upon reperfusion.

Ratio of von Willebrand Factor Propeptide to ADAMTS13 Is Associated With Severity of Sepsis

Von Willebrand factor (VWF)-cleaving protease (ADAMTS13) cleaves ultralarge VWF (ULVWF) secreted from endothelium and by which is regulating its physiologic function. An imbalance between ULVWF secretion and ADAMTS13 level occurs in sepsis and may cause multiple organ dysfunction. We evaluated the association between the VWF-propeptide (VWF-pp)/ADAMTS13 ratio and disease severity in patients with severe sepsis or septic shock. In 27 patients with severe sepsis or septic shock and platelet count less than 120,000/μL, we measured plasma VWF, VWF-pp, and ADAMTS13 levels on hospital days 1, 3, 5, and 7. The VWF-pp/ADAMTS13 ratio was increased greater than 12-fold in patients with severe sepsis or septic shock on day 1 and remained markedly high on days 3, 5, and 7 compared with normal control subjects. The VWF-pp/ADAMTS13 ratio significantly correlated with Acute Physiology and Chronic Health Evaluation II score on days 1 and 5; Sepsis-related Organ Failure Assessment score on days 1, 3, and 5; maximum Sepsis-related Organ Failure Assessment score and tumor necrosis factor α level on days 1, 3, 5, and 7; and creatinine level on days 1, 5, and 7. Patients with greater than stage 1 acute kidney injury had significantly higher VWF-pp/ADAMTS13 ratio than patients without acute kidney injury. In summary, the VWF-pp/ADAMTS13 ratio was associated with disease severity in patients with severe sepsis or septic shock and may help identify patients at risk for multiple organ dysfunction by detecting severe imbalance between ULVWF secretion and ADAMTS13 level.

VWF propeptide and ratios between VWF, VWF propeptide, and FVIII in the characterization of type 1 von Willebrand disease

AbstractDuring posttranslational modifications of von Willebrand factor (VWF), the VWF propeptide (VWFpp) is cleaved. The ratio between VWFpp and VWF antigen (VWF:Ag) and the ratio between factor VIII (FVIII:C) and VWF:Ag may be used to assess synthesis and clearance of VWF. We analyzed the contribution of VWFpp and ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag in the pathophysiological characterization of type 1 von Willebrand disease (VWD) in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD) study. The VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were increased among patients compared with unaffected family members and healthy controls. The VWFpp/VWF:Ag ratio was higher in individuals heterozygous for missense mutations than in those heterozygous for null alleles. In contrast, the FVIII:C/VWF:Ag ratio was highest among heterozygotes for VWF null alleles. The ratios of VWFpp/VWF:Ag and FVIII:C/VWF:Ag indicate that the pathophysiological mechanisms of type 1 VWD include reduced production and accelerated clearance of VWF, but that often a combination of both mechanisms is implicated.

Increased von Willebrand Factor Propeptide Levels in Symptomatic Versus Asymptomatic Moderate and Severe Carotid Artery Stenosis – Results from the Platelets and Carotid Stenosis (PACS) Study (S42.007)

Increased von Willebrand Factor Propeptide Levels in Symptomatic Versus Asymptomatic Moderate and Severe Carotid Artery Stenosis – Results from the Platelets and Carotid Stenosis (PACS) Study (S42.007)

Chronic dysfunction of the endothelium is associated with mortality in acute coronary syndrome patients

IntroductionPlatelet activation and endothelium dysfunction are determinants of atherothrombosis in acute coronary syndrome (ACS) patients. The aim of this study was to investigate the relationship between platelet and endothelial cell activation markers and mortality in patients presenting with ACS. Materials and MethodsPlasma levels of RANTES, Neutrophil Activating Protein-2 (NAP-2), Thrombospondin-1 (TSP-1), Von Willebrand Factor (VWF), Von Willebrand Factor Propeptide (VWF:pp) and Osteoprotegerin (OPG) were measured in a cohort study of 339 consecutive ACS patients who underwent percutaneous coronary interevention (PCI). The primary endpoint was 4-year mortality. ResultsThere were 46 deaths during the follow up. Median values of VWF (12.2μg/mL versus 7.86μg/mL, P=0.001) and VWF:pp (7.34nM versus 6.17nM, P=0.011) were higher in non-survivors compared to survivors. High levels of OPG were found in 37 patients: 27 of them were survivors (9.2%) and 10 were non-survivors (21.7%, P=0.011). Kaplan-Meier estimates of mortality for VWF were 7.5% in the first quartile (n=6 deaths), 12.2% in the second quartile (n=10 deaths), 11.2% in the third quartile (n=9 deaths) and 25% in the fourth quartile (n=21 deaths) of VWF (P=0.004). There was a 27.8% of probability of mortality when high OPG was measured versus 12.4% when low OPG was measured (P=0.007). No relationship between baseline platelet activation markers and mortality was found. ConclusionIn patients with ACS undergoing PCI, increased chronic endothelial cell activation and dysfunction is associated with an increased risk of long-term mortality.

Behavior of ADAMTS13 and Von Willebrand factor levels in patients after living donor liver transplantation

IntroductionThrombotic microangiopathy (TMA) is one of the important complications occurring after liver transplantation (LT), and it is suggested that a Von Willebrand factor (VWF) and ADAMTS13 may play an important role in the onset of TMA and poor outcome after LT. Materials and MethodsIn 81 patients after living donor LT (LDLT), 17 patients who had both severe thrombocytopenia and hemolytic anemia with fragmented red cell were diagnosed as TMA- like syndrome (TMALS) and 10 patients died. ResultsADAMTS13 activities were slightly low, and plasma levels of VWF and VWF propeptide (VWFpp) antigens and the ratio of VWFpp/VWF were significantly high before LDLT. ADAMTS13 activities were significantly reduced from day 1 to day 28 after surgery, and plasma levels of VWF antigen slightly decreased on day 1 and plasma levels of VWFpp continued to be high. The ratio of VWFpp/VWF was significantly high on day 1 after surgery.The mortality was high in the patients with TMALS and the frequency of TMALS was high in non-survivors. VWF levels were significantly low and the ratio of VWFpp/VWF was significantly high in those with TMALS on day 1 after surgery. The ADAMTS13 activity was significantly low, and the VWFpp and the VWFpp/ADAMTS13 ratio were significantly high in non-survivor on day 28 after surgery. ConclusionThese findings suggest that VWF and ADAMTS13 might therefore play an important role in the onset of TMA and poor outcome after LT. The VWFpp may therefore be a more useful marker for the diagnosis of TMALS than VWF.

Diagnosis of von Willebrand disease in Argentina: a single institution experience

Background: von Willebrand disease (VWD) is the most common autosomal bleeding disorder, mostly inherited as dominant trait. VWD is due to deficiency/abnormality of von Willebrand factor (VWF). The true prevalence of VWD is unknown, but estimated as 0.1% to 1% of the general population. Methods: The bleeding score (BS) was used to evaluate the bleeding status of patients presenting at our institution. Laboratory analyses include: VWF:Ag, VWF:RCo, VWF:CB, multimeric pattern, VWF propeptide, Desmopressin challenge test. Genotypic analysis comprises the study of specific exons of VWF, depending on the suspected variant. Results: We describe the main characteristics of our VWD population, including phenotypic and genotypic methods used to achieve the diagnosis. From our 2,482 patients registered, quantitative variants account for 83.2% of cases, and qualitative, 16.8% of cases. Platelet-type VWD (PT-VWD) was diagnosed in two patients and acquired von Willebrand syndrome (AVWS) in six. From quantitative variants, relative frequencies are: possible type 1, 78.7%; type 1, 14.9%; type 1S, 1.5%; type 1C, 0.4%; type 3, 1.6%. From qualitative variants: 2M, 21.9%; 2B, 10.3%; 2A, 8.4%; 2N, 3.4%; patients currently without complete diagnosis: 56%. Family history of bleeding: 64.7% of cases. Type 3, 2A and 2B VWD showed the highest major bleeding (MB) frequency. Genotypic diagnosis was reached in eight type 1C, 72 type 2M, 38 type 2B, 30 type 2A, 14 type 2N, and two PT-VWD patients. Five de novo mutations were identified among 52 families (9.61%): two mutations were associated with type 2B VWD (p.V1316M and p.S1310F), one mutation associated with type 1C (Vicenza) (p.R1205H) described in combination with p.R924Q, one deletion associated to type 1 VWD according to the ISTH VWF database (p.Pro1648fs*45) in combination with p.R1426C resulting in a 2M phenotype and one mutation related to type 2A VWD (p.Y1542D). Six patients are under prophylactic treatment due to their severe bleeding: four are type 3 VWD, one type 2M VWD, and one, the patient with p.Pro1648fs*45 and p.R1426C. Conclusions: The wide heterogeneity of clinical symptoms and laboratory data from possible type 1 VWD implies a risk for bleeding that becomes crucial in challenging situations. In our population, type 2M was found to be more frequent than the type 2A variant. DDAVP (1-deamino-8-D-arginine vasopressin) challenge test, VWF propeptide/antigen ratio and genotypic studies are useful tools for discriminating cases representing difficult diagnoses such as type 2M versus type 1C VWD. Elevated propeptide ratio, the detection of antiphospholipid antibodies, platelet-associated antibodies or anti-VWF immunoglobulins and negative genotypic screening is useful to discriminate inherited VWD from AVWS. Genotypic testing assisted discrimination between types 2B versus platelet-type-VWD and type 2N VWD versus mild hemophilia A.

Elevated Plasma Levels of Soluble Platelet Glycoprotein VI (GPVI) in Patients with Thrombotic Patients

AbstractAbstract 5145 IntroductionThrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets and it must be diagnosed at the early stage. This study, examined the activation of platelets by measuring of soluble platelet glycoprotein VI (sGPVI) level in patients with TMA. Materials and MethodsThe plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in 40 healthy volunteers, 46 patients without thrombosis (TH), 15 postoperative patients, 13 with disseminated intravascular coagulation (DIC) and 70 with TMA. The plasma levels of GPVI and VWFpp were measured by ELISA and ADAMTS13 was measured by the FRETS assay. There were 27 TMA with markedly decreased ADAMTS13 levels (TMA-ADAMTS13) and 43 TMA without (TMA-Other). ResultsThe plasma levels of sGPVI (median; 25. 0–75. 0%tile) were significantly higher in patients without TH (16. 2 ng/mL 12. 6–22. 5 ng/mL), postoperative patients (31. 6 ng/mL; 28. 3–35. 1 ng/mL), DIC (44. 5 ng/mL; 36. 6– 60. 8 ng/mL), TMA (40. 8 ng/mL; 32. 9–56. 7 ng/mL) than in healthy volunteers (11. 4 ng/mL; 9. 1–14. 8 ng/mL). In addition, the plasma levels of sGPVI post in postoperative patients were significantly higher than in patients without TH (p< 0. 001), and these levels were also significantly higher in those with TMA and DIC than in without TH (p< 0. 001, respectively). The plasma levels of sGPVI were significantly higher in patients with TTP-ADAMTS13 (36. 8 ng/mL; 30. 5–49. 0 ng/mL) and TMA-Other (51. 9 ng/mL; 37. 4–66. 3 ng/mL) than in patients without TH (p< 0. 001, respectively). ConclusionThe measurement of sGPVI is therefore considered to be important for the diagnosis of TMA. Disclosures:Wada:Mochida pharmaceutical CO., LTD: Suporting GPVI assay Other.

Genetic Determinants of Plasma Von Willebrand Factor (VWF) and VWF Propeptide Levels

Abstract 3313Plasma VWF levels vary by approximately five-fold in healthy populations and are influenced by both environmental and inherited factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis. Increased levels of VWF occur with age, may rise acutely due to inflammation or infection, and may serve as a biomarker for endothelial dysfunction and atherosclerosis. Genetic factors are thought to account for 60 – 70% of the variance in VWF antigen levels with ∼30% of this effect due to ABO blood type. VWF multimers are assembled in the Golgi apparatus where the VWF propeptide is cleaved to form mature VWF. Because the half life of plasma VWF propeptide (ppVWF) is ∼5X faster than the mature VWF antigen, the ratio of ppVWF/VWF:Ag in plasma primarily reflects clearance rates of mature VWF while alterations in biosynthesis and/or secretion of VWF affect the level of both ppVWF and VWF:Ag. In order to identify additional quantitative trait loci (QTL) for VWF antigen variation and altered clearance of VWF:Ag, we performed genetic analyses in a healthy young sibling cohort, the Genes and Blood Clotting Study (GABC, n=1,152). The cohort was SNP genotyped with an Illumina Omni-1 array resulting in 763,195 SNPs available for analysis after extensive QC. Plasma VWF antigen (VWF:Ag) and VWF propeptide levels were determined by AlphaLISA (Perkin Elmer, Waltham, MA), using polyclonal anti-human VWF antibodies (DAKO, Glostrup, Denmark) or monoclonal anti-ppVWF antibodies (Blood Center of Wisconsin, Milwaukee, WI). The median levels were 108.1 IU/dL, 99.97 IU/dL and 1.0 for VWF:Ag, ppVWF and ppVWF/VWF:Ag respectively. The estimated heritability (h2) was 64.5%, 55.72%, and 64.26% for VWF:Ag, ppVWF, and ppVWF/VWF:Ag respectively. A genome-wide association study (GWAS) for VWF:Ag in this cohort revealed significant signals at the ABO and VWF loci, consistent with previously published reports. However, GWAS for ppVWF levels demonstrated a signal only at 12p13, overlying the VWF gene itself. The top SNP, rs1800378 (beta 4.297, p-value 1.58E–11), encodes a nonsynonymous substitution (His484Arg) in the VWF propeptide. In contrast, GWAS for ppVWF/VWF:Ag showed a strong signal at 9q34, the ABO locus, (top SNP rs687289, beta −0.2131, p-value 1.88E–62) and no significant signal at 12p13. These results suggest that ABO is the major common determinant of variability in VWF clearance rates and a common SNP, rs1800378, is associated with VWF propeptide levels. We next utilized the sibling structure in this cohort to perform linkage analyses. Linkage analysis for VWF:Ag identified a novel locus at 2p12-2q13 in addition to the expected signal at ABO. The effect size of this chromosome 2 locus on VWF variation (19.2% variance explained) was comparable to the effect of the ABO locus (24.5%). Linkage analysis for ppVWF demonstrated novel loci at 7q21 (LOD score 5.03) and at 11q21 (LOD score 4.12), with no significant signal on chromosome 2. In contrast, linkage analysis for ppVWF/VWF:Ag identified signals at 2p12 (LOD score 3.17), 8p22 (LOD score 3.48) and 11q22 (LOD score 3.41). Taken together, these results identify a number of potential candidate loci for genetic regulation of plasma VWF through alteration of synthesis, storage, secretion or clearance mechanisms. We hypothesize that signals present in linkage but not association are due to VWF regulatory genes with high allelic heterogeneity, harboring many genetic variants that are individually rare, but in aggregate present in a large portion of sibships. Segregation of these variants among sibs could then explain the strong signal by linkage, but lack of signal by association in unrelated individuals. Replication of the strong 2p12-2q13 signal in both VWF:Ag and ppVWF/VWF:Ag linkage studies suggests that the responsible variants at this locus affect VWF levels through altered clearance. The novel QTLs for ppVWF/VWF:Ag levels on chromosome 8p22 and 11q22 are also likely to work through altered clearance mechanisms while the novel signals for ppVWF levels on 7q21 and 11q21, not significant in the VWF:Ag linkage study, may affect efficiency of cleavage or clearance of ppVWF. Identification of the underlying genes at these novel loci and their mechanism of action could lead to improved understanding of the severity, penetrance and treatment for von Willebrand Disease as well as the risk for venous thromboembolic disease associated with elevated VWF. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Distinct Functions of pH-Sensing Histidine Residues in the Multimer Assembly and Storage of Von Willebrand Factor in Weibel-Palade Bodies

Abstract 3311The hemostatic function of von Willebrand factor (VWF) depends on the formation of disulfide-linked multimers. We have previously shown that several evolutionarily conserved His residues within the VWF propeptide are critical for pH-dependent VWF multimerization. For example, the charge-neutralization mutations H395A and H460A in the VWF D2 domain prevented multimer assembly, while the charge-stabilization mutation H460K preserved it, suggesting that protonation at these sites is essential. In this study, we investigated the role of these His residues in VWF targeting to storage granules and agonist-induced secretion. Full-length VWF was expressed in transiently-transfected HEK293 cells. If Weibel-Palade body-like storage granules (WPBs) were formed, their morphology was analyzed by immunofluorescence microscopy. A WPB was defined as “long” if length/width was > 2, or “round” if length/width was ≤ 2. To analyze the assembly of VWF multimers, we stimulated VWF secretion by incubating cells with 100 ng/ml of phorbol myristate acetate for 1 hour and collected cell lysates and supernatants for multimer gel electrophoresis. Expression of wild type VWF and VWF H460K induced the formation of approximately 60% long WPBs. Treatment of cells with monensin increases the pH of the Golgi, and as expected monensin reduced the storage of wild type VWF in long WPBs. However, monensin did not impair the storage of VWF H460K or reduce the fraction of long WPBs, indicating that a positive charge at residue 460 can sustain tubular storage of VWF despite neutralization of the late secretory pathway. Both WT VWF and VWF H460K were secreted with a normal distribution of multimers. VWF H395A and VWF H460A formed predominantly round pseudo-WPBs, with only 10–20% long granules, and these mutations also disrupted VWF multimerization. In contrast, VWF H395R and VWF H395A/H460R did not assemble multimers but nevertheless formed long WPBs. Insertion of an extra Gly between the vicinal cysteines in the D1 domain (159CGLC162 to 159CGGLC162) was reported to prevent multimerization but allow storage in long WPBs in AtT-20 cells (Mayadas & Wagner, PNAS 1992; 89:3531). We found that this mutation also inhibited VWF multimerization and resulted in the formation of long WPBs in HEK293 cells. These results indicate that VWF multimerization and packing in elongated WPBs both depend on the acidic pH of the late secretory pathway. However, these processes are independent, with distinct structural requirements, and they can be dissociated by mutations of specific His residues.VWF constructMultimersGranule ShapeGranule Shape (with monensin)Wild typeYesLongRoundH395ANoRoundH395RNoLongH395A/H460RNoLongH460ANoRoundH460KYesLongLong159CGGLC162NoLong Disclosures:No relevant conflicts of interest to declare.

Biogenesis and Exocytosis of Weibel-Palade Bodies Is Affected by Naturally Occurring Von Willebrand Disease Variants within the A1-A3 Domains of VWF

AbstractAbstract 1072 Background:Von Willebrand Factor (VWF) is synthesized in endothelial cells and megakaryocytes and is either secreted constitutively into plasma or stored in specific organelles; Weibel-Palade bodies (WPB) in endothelial cells or α -granules in megakaryocytes and platelets. Release of von Willebrand factor from WPB in response to desmopressin (DDAVP), an agonist of WPB exocytosis, is clinically applied to raise VWF plasma levels in patients with von Willebrand disease (VWD). A subset of patients with VWD type 1, a quantitative defect, shows a reduced response to DDAVP. This reduced response suggests the absence of recruitable WPB. The VWF propeptide (D1-D2 domains) together with the D’D3 domains are necessary for the tubular assembly of VWF. The assembly of VWF into tubules drives WPB formation. Some variants in the VWF A1 to A3 domains have been linked to reduced DDAVP responsiveness. This suggests that determinants for WPB formation may reside outside the D1-D2-D’-D3 domains. We hypothesize that a reduced tendency to assemble into VWF tubules underlies the defective DDAVP response in VWD type 1 patients with mutations in the A domains of VWF. Methods:Human Embryonic Kidney (HEK) 293 cells were transiently transfected with plasmids containing full-length wild-type VWF or 6 naturally occurring VWF variants located throughout the A domains of VWF. The following mutations, originally identified in type 1 VWD patients, were studied: p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His (A1 domain), p.Tyr1584Cys, p.Arg1583Trp (A2 domain) and p.Val1822Gly (A3 domain). Medium and lysates of transfected cells were collected to measure basal VWF secretion. Transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the regulated VWF secretion. Confocal- and Transmission electron microscopy (TEM) were used to study the formation of WPB. Results:Cells transfected with WT-VWF or VWF p.Arg1583Trp formed numerous elongated pseudo-WPB as evidenced by confocal microscopy. p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His, p.Tyr1584Cys and p.Val1822Gly were able to form these organelles, but in most cases the pseudo-WPB were shorter and more round compared to those formed by WT-VWF. Retention of VWF in the ER was present in about 50% of the cells expressing p.Leu1307Pro and 25–35% of the cells expressing p.Ser1285Pro and p.Val1822Gly as compared to 10% in WT-VWF. The ER retention in p.Arg1374His, p.Tyr1584Cys and p.Arg1583Trp variants was comparable with WT-VWF. Upon co-transfection with WT-VWF the defective elongation of pseudo-WPB was partly corrected. WPB formation was further studied using TEM. In WT-VWF transfected cells, elongated and electron dense pseudo-WPB were observed. p.Arg1583Trp showed cigar-shaped pseudo-WPB with typical VWF striations. Shorter and more round pseudo-WPB were found in cells expressing the VWF variants p.Ser1285Pro, p.Arg1374His, p.Tyr1584Cys. The p.Val1822Gly variant showed some elongated pseudo-WPB, although most of the structures were round. The round organelles are, however, recognizable as pseudo-WPB as they contain tubular structures indicating storage of VWF tubules. The p.Leu1307Pro and p.Val1822Gly showed reduced VWF basal secretion, even in the heterozygous state. Both mutations, together with p.Ser1285Pro, also showed impaired regulated secretion of VWF in both single and co-transfections with wt-VWF. Conclusion:Our data shows that naturally occurring VWD variants within the A domains of VWF can cause defects in both WPB formation and (regulated) secretion of VWF. This is in contrast with previous data which suggests that only the propeptide and D1'-A1 domain of VWF are essential for normal WPB formation. In our study however, we found that two mutations located within the A2 and A3 domain (p.Tyr1584Cys and p.Val1822Gly) also interfere with the formation of elongated WPB as evidenced by round storage organelles containing VWF tubules. Defects in WPB formation and regulated secretion of VWF may be the underlying cause of the poor response to DDAVP infusion seen in a subset of VWD type 1 patients. Disclosures:No relevant conflicts of interest to declare.

Imaging of Von Willebrand Factor Remodeling Upon Secretion From Vascular Endothelial Cells

AbstractAbstract 263In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation.To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell.In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures:No relevant conflicts of interest to declare.

Intracellular Retention, Enhanced Clearance, and Defective FVIII Binding Are Common Features of Von Willebrand Factor D'-D3 Domain Mutations in Patients with Von Willebrand Disease Type 1 From the European Mcmdm-1VWD Study

AbstractAbstract 99 Background:Von Willebrand disease (VWD) type 1 is characterized by a partial reduction of structurally and functionally normal VWF with normal VWF multimers. As part of a large European study (Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) patients previously diagnosed with VWD type 1 were studied systematically to assess the phenotypic and genotypic spectrum. Objective:To confirm the pathogenicity of VWF gene mutations and to elucidate the molecular mechanisms of VWD type 1. Patients and methods:VWD type 1 patients were recruited by twelve expert centers in nine European countries. VWF genotyping was performed in all index cases (IC). The eight mutations studied here are located in the VWF D'-D3 domain and corresponded to 57 patients from 19 families. They were reproduced by recombinant expression with subsequent phenotypic characterization, two of them in cis and one in trans with a second mutation. Results and Discussion:Intracellular VWF:Ag of all mutants was normal or near normal suggesting normal expression levels. However, seven mutations (p.M771I, p.I1094T, p.C1130R, p.C1130G, p.C1130F, p.W1144G and p.Y1146C) caused intracellular retention and impaired VWF secretion. In addition, we observed a major loss of high molecular weight multimers as in type 2A and a novel finding of a severe VWF:FVIII binding defect in most of the homozygously expressed mutants. Additional mutations either in cis or in trans had no modifying effect. The recombinant VWD type 1 Vicenza mutation p.R1205H with or without the allelic variant p.M740I seen in three Italian IC was secreted normally and had normal function leaving enhanced clearance of mutant VWF as the only pathomechanism. In conclusion, the majority of mutations in the D3 domain impair VWF multimerization, cause intracellular retention and correlate with defective FVIII binding. An elevated ratio of VWF propeptide to VWF:Ag suggests enhanced VWF clearance as an important pathomechanism of most mutations and particularly of p.R1205H. Disclosures:No relevant conflicts of interest to declare.

Left Ventricular Assist Device–Induced Coagulation and Platelet Activation and Effect of the Current Anticoagulant Therapy Regimen

Left ventricular assist devices (LVADs) are mechanical pumps that enhance cardiac function in patients with heart failure. In all, 7 patients with an LVADs (1.8 international normalized ratio warfarin, 81 mg aspirin) were evaluated monthly for 3 months for platelet and coagulation activation (controls: 5 healthy adults and 5 patients having warfarin). Platelet works revealed greater inhibition of collagen (31.8% vs 7.9%; P = .004), arachidonate- (30.9% vs 8.2%; P = .001), and adenosine diphosphate- (10.9% vs 6.1%; P = .004)-induced platelet aggregation for LVADs. Thrombelastography (recalcified whole blood) showed inhibition of clot initiation time (R; 8.81 vs 6.02 min; P = .001) and stronger clot formation (maximum amplitude; 69.1 vs 64.9 mm; P = .016). Platelet function determined by plateletMapping and flow cytometry was within the normal range. The LVADs had increased ratio of von Willebrand Factor (vWF) antigen and vWF propeptide, indicating increased degradation of vWF (2.04 vs 1.44; P = .144). Coagulation and platelet activation caused by LVAD is suppressed by pharmacotherapy, yielding a profile similar to that of patients on warfarin alone.

Von Willebrand factor (VWF) propeptide binding to VWF D′D3 domain attenuates platelet activation and adhesion

AbstractNoncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D′D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D′D3 domain. At pH 6.2 and 10mM Ca2+, conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (KD = 0.2nM, koff = 8 × 10−5 s−1). Significant, albeit weaker, binding (KD = 25nM, koff = 4 × 10−3 s−1) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca2+. This interaction was also observed in human plasma (KD = 50nM). The addition of recombinant VWFpp in both flow-chamber–based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D′D3 mAb DD3.1, which blocks VWFpp binding to VWF-D′D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.