Von Willebrand Factor Levels In Patients Research Articles

Von Willebrand Factor Dynamics in Patients with Aortic Stenosis Undergoing Surgical and Transcatheter Valve Replacement.

Aortic stenosis (AS) is a prevalent valvular disorder that poses a significant burden on healthcare systems due to its debilitating symptoms and high mortality rates if left untreated. Surgical aortic valve replacement (SAVR) and transcatheter aortic valve replacement (TAVR) are the primary interventions for severe AS, but perioperative complications such as bleeding remain a concern. Von Willebrand factor (VWF), a crucial player in hemostasis, is known to be altered in AS and may contribute to the hemostatic imbalance observed in these patients. This prospective study aimed to investigate the association between prosthetic valve type, size, and postprocedural VWF levels in patients undergoing aortic valve replacement (AVR) for severe AS. This study involved 39 consecutive patients diagnosed with severe AS who underwent SAVR or TAVR. By elucidating the VWF dynamics associated with different prosthetic valves, this study sought to provide valuable insights into personalized valve selection and perioperative management strategies.

Effect of eplerenone on aldosterone and von Willebrand factor levelsin patients with myocardial infarction and chronic kidney disease

Aim: to study the levels of aldosterone and von Willebrand factor (VWF) in patients with Q-wave acute myocardial infarction (Q-AMI) and underling chronic kidney disease (CKD) using aldosterone antagonists. Materials and methods. The study group consisted of 106 patients with renal dysfunction who were hospitalized for acute Q-AMI. Serum levels of neurohumoral parameters (aldosterone and VWF) were determined in all the patients by enzyme-linked immunosorbent assay kits. Results. Reduction in the aldosterone levels was found in patients with Q-AMI and stage 2 CKD from 245.08 ± 17.38 pmol/l to 195.15 ± 13.82 pmol/l (P < 0.05;) who received spironolactone and from 275.59 ± 23.43 to 169.37 ± 24.46 pmol/l (P < 0.001) with eplerenone usage. Patients with stage 1 CKD showed a decreasing trend in aldosterone levels when using spironolactone (from 238.04 ± 20.37 pmol/l to 200.78 ± 9.15 pmol/l), and significantly decreased aldosterone levels when receiving eplerenone (from 229.77 ± 13.76 pmol/l to 156.76 ± 5.76 pmol/l; P < 0.05). The serum concentration of VWF in stage 2 CKD patients was changed insignificantly on spironolactone therapy (from 0.99 ± 0.13 mg/l to 1.13 ± 0.06 mg/l) and it was significantly decreased in the eplerenone group – from 1.29 ± 0.19 mg/l to 0.71 ± 0.14 mg/l (P < 0.05). In stage 1 CKD patients, the concentration of VWF was not decreased significantly with the use of spironolactone (from 1.22 ± 0.13 mg/l to 1.03 ± 0.06 mg/l), but it was decreased significantly when receiving eplerenone (from 1.14 ± 0.09 mg/l to 0.79 ± 0.08 mg/l; P < 0.05). The data obtained indicate the significantly higher probability of declining both aldosterone (t = 2.91; P < 0.01) and VWF (t = 2.59; P < 0.01) with the use of eplerenone in the complex treatment as compared to the spironolactone administration. Conclusions. The effect of eplerenone to decrease the concentration of aldosterone and VWF is significantly greater than that of spironolactone, especially in AMI patients with stage 2 CKD.

Bleeding Outcomes in People with Von Willebrand Disease Receiving Antithrombotic Therapy

▪Aging brings additional challenges in the management of people with von Willebrand Disease (VWD). Plasma von Willebrand Factor (VWF) levels may increase but the impact on bleeding phenotype is unclear. With the development of age-related comorbidities, the use of antiplatelet (AP) or anticoagulant (AC) therapies may be warranted. As highlighted in the 2021 international VWD guidelines, limited evidence exists regarding the bleeding risk and safety of AP/AC use in people with VWD. 1 We sought to address this knowledge gap through a retrospective review of a large cohort of people with VWD attending a tertiary referral center.The records of all patients aged >50 years (y) attending our center registered with VWD were retrospectively reviewed. We identified all individuals treated with AP and/or AC, recording the indication for and duration of therapy. We also recorded disease subtype, baseline and most recent plasma VWF levels and bleeding on AP and/or AC. Bleeding episodes were stratified according to the World Health Organization (WHO) Bleeding Scale.From 255 eligible patients, 18 patients (7 male, 11 female) were identified who received AP and/or AC over a period of 22y. The median age at commencement was 60.2 years (range 40.0-74.5). 15 patients had Type 1 with baseline levels 30-50 IU/dL, (median VWF antigen, VWF:Ag, 57.5, range 39-87 IU/dL; median VWF ristocetin cofactor levels, VWF:RCo, 43, range 35-54 IU/dL). 3 patients had type 2 VWD (median VWF:Ag 64, range 27-90 IU/dL; median VWF:RCo of 16, range 10-66 IU/dL). The type of AP/AC used and indications for treatment are outlined in Table 1.Overall, 12 patients were treated with AP and 7 with AC therapy (19 therapies in total as one patient received first aspirin then warfarin therapy). The cumulative exposure to AP therapy was 61.9y with a median exposure time of 3.2y/patient (range 0.3-14.1y). Duration of AC therapy was shorter, with a cumulative exposure of 17.6y and a median of 1.5 y/patient (range 0.3-6.3y).Overall, 85.7% of patients on AC therapy had at least one episode of bleeding (6/7; 10 episodes total) in contrast to 58.3% of patients on AP (7/12; 12 episodes total). Of these 22 episodes, 5 (22.7%) were grade 1 bleeding. Grade 2 bleeding (iron deficiency or gastrointestinal (GI), gynecological or genitourinary bleeding) occurred in 5 patients (41.7%) treated with AP and 4 patients (57.1%) on AC (total of 14 episodes). 1 episode of grade 3 bleeding occurred in both the AP (8.3%) and AC (14.3%) group (GI bleeding requiring transfusion and abdominal hematoma respectively). The single grade 4 bleed was an intracranial hemorrhage (ICH) and occurred in a patient with type 2 VWD (VWF:RCo 10 IU/dL) on warfarin for atrial fibrillation; this required prothrombin complex concentrate, VWF concentrate, neurosurgical intervention and cessation of AC.Bleeding complications resulted in discontinuation of therapy in 2 patients (11.1%) treated; the individual with ICH and a patient with type 1 VWD on warfarin (baseline VWF:RCo 43 IU/dL) due to recurrent GI bleeding. No patients treated with AP therapy required discontinuation of use. The overall rate of major bleeding (WHO grade >/=3) in our study was 11.4 events/100 patient-years in VWD patients receiving AC therapy, in comparison to the rate of bleeding in the general population using AC of 7.2 events per 100 patient-years. 2For patients with type 1 VWD, plasma VWF levels were seen to increase during follow up (median 8.5y, VWF:Ag median +13 IU/dL, VWF:RCo +18 IU/dL), resulting in plasma VWF levels >50 IU/dL for 66.6% of patients in this cohort. Despite this, bleeding while on AP and/or AC was still experienced in 8/10 patients whose levels had normalized, necessitating cessation in one instance.In conclusion, this study provides important insights into the use of AP and/or AC in patients with VWD. Bleeding rates were higher in patients treated with AC therapy than AP resulting in cessation of therapy in 28.6% of those on AC. Bleeding events still occurred despite normalization of plasma VWF levels in patients with type 1 VWD. These data highlight the need for close follow up of patients with VWD whilst on antithrombotic therapy, particularly AC.1. Connell NT et al. ASH ISTH NHF WFH 2021 guidelines on the management of von Willebrand disease. Blood Adv 2021;5(1):301-325.2. Shoeb M, Fang M. Assessing Bleeding Risk in Patients Taking Anticoagulants. J Thromb Thrombolysis 2013;35(3):312-319. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Raised von Willebrand Factor Levels in Liver Diseases: Opening Up Newer Therapeutic Avenues

Raised levels of the blood coagulation protein von Willebrand factor (VWF) are now recognised to be important in patients with liver disease. The markedly raised plasma VWF levels in patients with acute liver failure and acute-on-chronic liver failure may contribute to the pathogenesis of liver failure, and of multi-organ failure, by impeding microcirculatory perfusion in the liver and the other affected vital organs. In this review, the authors present a brief introduction to VWF biology, discuss the ability of raised plasma VWF levels to accurately predict survival in different syndromes of liver diseases, speculate why plasma VWF levels are raised in liver failure syndromes, and examine the therapeutic potential of VWF-lowering therapies in these scenarios.

Novel Insights into the Clinical Phenotype and Pathophysiology Underlying Low VWF Levels: The Low Von Willebrand Factor in Ireland Cohort (LoVIC) Study

▪Cohort studies have identified VWF gene mutations in ~65% of patients with type 1 Von Willebrand Disease (VWD). However, in patients with mild reductions in Von Willebrand Factor (VWF) levels, VWF mutations are less common and linkage studies suggest that the reduced plasma VWF levels are often independent of the VWFgene. Important clinical questions therefore remain unanswered regarding diagnosis and management of patients with Low VWF (levels 30-50 IU/dL). Evidence-based diagnostic criteria are needed for establishing the diagnosis of Low VWF, and the relationship between Low VWF levels and bleeding phenotype needs to be defined. Furthermore, the molecular mechanisms responsible for the reduced plasma VWF levels remain unclear. To address these questions, we established the Low VWF Ireland Cohort (LoVIC) study. In contrast to previous Type 1 VWD studies, this prospective longitudinal cohort study recruited only patients with Low VWF levels, (defined by bleeding history and lowest VWF levels 30-50 IU/dL on two occasions, three months apart) and recruited 140 adult Irish patients.Although Bleeding Assessment Tools (BATs) have been used in type 1 VWD, their utility has not been studied in patients with Low VWF. As determined using either the ISTH BAT or the Condensed MCMDM-1 VWD score, we observed significant bleeding histories in the majority of LoVIC patients. For example, among female patients (n=112), 77% had positive ISTH BAT scores (≥6) and 72% had positive MCMDM-1 scores (≥4). Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30-50 IU/dL range. To further investigate this bleeding phenotype, hemostatic studies (including platelet aggregation and coagulation factor assays) were performed. Additional mild coagulation defects were identified in only 10 subjects. Furthermore, abnormal multimer patterns were identified in only 3 patients. To investigate the pathophysiology underlying Low VWF levels, plasma FVIII:C and VWF propeptide (VWF:pp) levels were defined. Interestingly, plasma FVIII:C/VWF:Ag ratios were significantly increased in LoVIC patients compared to normal controls (mean 1.3 versus 1.07; p<0.001). In contrast, increased plasma VWF:pp/VWF:Ag ratios > 3 were observed in only 6% of the total cohort. Taken together, these data demonstrate that the reduced plasma VWF levels in patients with Low VWF are predominantly attributable to decreased VWF synthesis and/or secretion rather than enhanced VWF clearance. To determine whether quantitative and/or qualitative abnormalities in platelet (plt)-VWF may influence bleeding phenotype in patients with Low VWF, we studied plt-VWF:Ag and VWF:CB in 50 consecutive LoVIC patients. In keeping with the hypothesis that reduced VWF synthesis plays a key role in the pathogenesis underlying Low VWF levels, plt-VWF:Ag and plt-VWF:CB levels were both significantly reduced in LoVIC patients compared to controls (mean plt-VWF:Ag 0.16 versus 0.21 IU/109/L, p<0.05; mean plt-VWF:CB 0.18 versus 0.34 IU/109/L, p<0.001).To further study the molecular mechanisms underlying Low VWF levels, plasma VWF:Ag and VWF:RCo responses following desmopressin (DDAVP) administration were examined. In 88% of patients, the post-DDAVP peak levels exceeded 100 IU/dL. Importantly, the response to DDAVP was also sustained with both VWF:Ag and VWF:RCo above 100 IU/dL after 4 hours in 72% subjects. These DDAVP responses demonstrate that Weibel Palade body stores of VWF are maintained in patients with Low VWF, and that the regulated pathway of VWF secretion is intact. Furthermore, the sustained plasma VWF response observed following DDAVP supports the hypothesis that enhanced VWF clearance does not play a major role in the pathogenesis of Low VWF levels.In conclusion, we demonstrate that Low VWF levels are associated with a significant bleeding phenotype. In addition, we further show that in the majority of patients with Low VWF levels, the bleeding phenotype cannot be explained by the presence of concomitant bleeding disorders. Finally, our novel data demonstrate that Low VWF levels are due in large part to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in a minority of individuals with Low VWF, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. DisclosuresLavin:Baxalta: Consultancy, Research Funding. O'Sullivan:Pzifer: Research Funding. O'Connell:Baxalta: Consultancy, Research Funding. James:Octapharma: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen: Consultancy; Basalt: Consultancy. Di Paola:CSL BEhring: Consultancy; Biogen: Consultancy. O'Donnell:Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Leo Pharma: Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

Higher and lower active circulating VWF levels: different facets of von Willebrand disease.

Most circulating von Willebrand factor (VWF) is normally inactive and incapable of binding platelets, but numerous disorders may modify the proportion of active VWF. We explored active VWF levels in patients with von Willebrand disease (VWD) whose VWF had a higher affinity for platelet glycoprotein (GP)Ib, but different susceptibilities to ADAMTS13 and multimer patterns (9 patients lacking large multimers, 10 with a normal pattern); 12 patients with VWF C2362F and R1819_C1948delinsS mutations, which make VWF resistant to ADAMTS13 were also studied. Type 2B patients with abnormal or normal multimers had significantly more active VWF (3·33 ± 1·6 and 3·74 ± 0·74, respectively; normal 0·99 ± 0·23). The type of VWF mutation influenced VWF activation: V1316M was associated with the highest levels in patients with abnormal multimers, and R1341W in those with normal multimers. Pregnancy induced gradually rising active VWF levels and declining platelet counts in one type 2B VWD patient without large multimers. Active VWF levels dropped significantly in patients homozygous for the C2362F mutation or heterozygous for R1819_C1948delinsS mutations (0·2 ± 0·03 and 0·23 ± 0·1, respectively), and less in cases heterozygous for the VWF C2362F mutation (0·55 ± 0·17). We demonstrate that VWF may be more or less activated, with or without any direct involvement of the A1 domain, and regardless of ADAMTS13.

CLEC4M and STXBP5 gene variations contribute to von Willebrand factor level variation in von Willebrand disease

von Willebrand factor (VWF) levels in healthy individuals are influenced by variations in genetic loci other than the VWF gene, whose contribution to VWF levels in patients with von Willebrand disease (VWD) is largely unknown. To investigate the association between single-nucleotide polymorphisms (SNPs), VWF levels, and bleeding phenotype. In 364 type 1 VWD and 240 type 2 VWD patients from the nationwide cross-sectional 'Willebrand in The Netherlands' (WiN) study, we studied the association between eight SNPs in STXBP5, SCARA5, ABO, VWF, STAB2, STX2, TC2N, and CLEC4M, and VWF antigen (VWF:Ag), VWF activity (VWF:Act), and bleeding phenotype as assessed with the Tosetto bleeding score. In type 1 patients, STXBP5 was associated with a lower VWF:Ag level (adjusted difference of -3.0 IU dL(-1) per allele; 95% confidence interval [CI] -6.0 to 0.1) and CLEC4M with both a lower VWF:Ag level (-4.3 IU dL(-1) per allele; 95% CI -7.9 to -0.6) and lower VWF:Act (-5.7 IU dL(-1) per allele; 95% CI -10.9 to -0.5). In type 2 patients, none of the SNPs was associated with VWF levels. None of the genetic variants was associated with bleeding score. Genetic variations in STXBP5 and CLEC4M are associated with VWF level variation in type 1 VWD, but not in type 2 VWD. This study increases our understanding of the pathophysiology of VWD, and provides a further indication of the involvement of STXBP5 and CLEC4M in determining VWF levels in VWD.

Mutations in the D1 Domain Inhibit Propeptide-Dependent Multimerization, Trafficking and Regulated Secretion of Von Willebrand Factor in VWD

Gene mutations play an important role in the pathogenesis of von Willebrand disease (VWD), resulting in the qualitative defect or quantitative deficit of von Willebrand factor (VWF). VWF propeptide is composed of D1 and D2 domain, which acts as a covalent oxidoreductase in the multimerization. In addition, the propeptide is necessary for the transport from endoplasmic reticulum (ER) to Golgi-apparatus, basal secretion and regulated secretion of VWF. However, the mechanisms of the mutation in D1 domain impairing VWF multimerization and causing low VWF levels in patients remain unknown. Herein we identified four mutations in the D1 domain from VWD patients, and assessed the effect of these mutations on the function of propeptide.We identified p.G39R, p.D141N, p.K157E, and p.C379G in three VWD patients. To characterize the roles of four mutations in propeptide-depend multimerization, we generated several truncated-VWFs, D1D2D¡¯D3 (residues 1-1241), including G39R, K157E, D141N, C379G and wide type (WT). These constructs were then expressed in HEK 293 cells, and were evaluated the D¡¯D3-dimer formation of mutations and WT. Full-length VWF comprising mutations and WT were also restructured and were transfected in HEK 293 cells. We then analyzed the VWF multimer distribution and VWF antigen in the cell supernatant and cell lysate. We also assessed VWF retention in ER and the stimulation secretion by phorbol-12-myristate-13- acetate (PMA) of mutations and those of WT.Two type 3 and 1 type 1 patients were enrolled in our study. VWF antigen were 3, 1 and 8 IU/dL, and VWF:Rco were 2.1, 2.3 and 5.6 IU/dL respectively in three patients. VWF multimer distribution exhibited none in two type 3 patients and normal-like multimer pattern in the type 1. Sequence analysis of VWF gene showed two heterozygous mutations (p.G39R and D141N) in D1 domain of one type 3 patient, and a heterozygous K157E in propeptide and a heterozygous C1165R in D3 domain of the other patient with type 3. Type 1 patients had a heterozygous C379G mutation. Among these four mutations in D1 domain, p.G39R, p.K157E, and p.C379G were novel.In the supernatant of transfected cell, dimerization of D¡¯D3 was absent in truncated-G39R. Compared with that of truncated-WT, decreased but detectable dimerizations were detected in K157E, D141N, and C379G. Similar results were also observed in multimerization of full-length constructs. The multimer assembly was too low to visualize in G39R, whereas decreased medium and absent large VWF multimers were seen in K157E, D141N, and C379G.By immunofluorescence imaging, all full-length VWF variants were detained in ER in different degrees (fig 1). The basal secretions of G39R, K157E, D141N, and C379G were (2.7¡À0. 3)%, (2.5¡À0.2)%, (26.0¡À4.1)%, and (22.4¡À3.8)% of WT respectively. However, VWF antigens in the lysate of transfected cells were (116.5¡À5.4)%, (90.9¡À3.0)%, (91.7¡À0.4)% and (113.8¡À2.9)% of WT for G39R, K157E, D141N, and C379G. No detectable secretion increases of mutant VWF induced by PMA were observed in the transfected cells, while WT-VWF with PMA exhibited increased secretion from 0.69% to 1.66%. [Display omitted] These four mutations in D1 domain downgraded the activity of propeptide as the covalent oxidoreductase, and impaired the muiltimeriztion induced by propeptide. They also interfered with VWF transport from ER to Golgi-apparatus and caused the VWF retentions in ER. Therefore, they further reduced the basal secretion and regulated secretion of mature VWF, which could explain the possible pathogenesis of quantitative deficit of VWF in VWD. DisclosuresNo relevant conflicts of interest to declare.

Relationship of Von Willebrand Factor with carotid artery and aortic arch calcification in ischemic stroke patients

BackgroundLarge population studies have revealed that increased von Willebrand Factor (VWF) levels are associated with an increased risk of ischemic stroke. In previous studies VWF was associated with atherosclerosis in healthy individuals. However, it is yet unknown what the association is between atherosclerosis and VWF levels in patients with ischemic stroke. ObjectivesThe aim of our study was to determine the association of atherosclerosis, measured with recent developed techniques, and VWF levels in a large, well characterized, cohort of ischemic stroke patients and to determine the prognostic value. MethodsWe included 925 consecutive patients with transient ischemic attack (TIA) or ischemic stroke. Calcification volumes (mm3) were scored in the aortic arch and both carotid arteries using multidetector computed tomography (CT) angiography. VWF antigen (VWF:Ag) levels were measured using ELISA. ResultsMean VWF:Ag levels were significantly higher in the presence of calcification in either the aortic arch (1.47 vs. 1.37 IU/ml [P = 0.039]) or the carotid arteries (1.49 vs. 1.34 IU/ml [P = 0.001]). Patients with a large artery atherosclerosis ischemic stroke had significantly higher VWF:Ag levels then the other TOAST subtypes (P < 0.0001). High VWF:Ag levels were associated with an unfavorable outcome (modified Rankin Scale >2 vs. ≤2; 1.64 vs. 1.41 IU/ml, [P < 0.0001]). ConclusionOur study showed a strong association between the extent of atherosclerosis in both the aortic arch and the carotid arteries and VWF levels in patients with TIA or ischemic stroke. Higher VWF levels are found in large artery atherosclerosis and are associated with a poor outcome.

Decreased ADAMTS13 Levels in Patients after Living Donor Liver Transplantation

Introduction Thrombotic microangiopathy (TMA) is a complication occurring after liver transplantation (LT), and an unusually large multimer (ULM) of Von Willebrand factor (VWF) and ADAMTS13 may play an important role in the onset of TMA during LT. Material and Methods Eight-one patients underwent living donor LT (LDLT). Seventeen of those patients had both severe thrombocytopenia and hemolytic anemia with fragmented red cells and were diagnosed as TMA- like syndrome (TMALS). Results and Conclusions A significant reduction of ADAMTS13 and an increase of VWF were observed in the patients with TMALS. The ADAMTS13 activity in patients after LDLT was significantly reduced from day 1 to day 21, and it was significantly low in those with TMALS at day 14 and 28. The VWF levels in patients with LDLT were significantly high, and the VWF/ADAMTS13 ratio was significantly increased in patients at 7, 14 and 28 days after LDLT, especially in patients with TMALS at day 14 and 28 after LDLT. High molecular weight multimers of VWF were observed to have increased in patients with LDLT, and the high molecular weight multimers of VWF were further increased in those with mild TMALS but they decreased in those with severe TMA. These findings suggest that ULM- VWF and ADAMTS13 might be associated with the onset of TMA after LT.

The dynamic changes of plasma yon willebrand factor and P-selectin in the finger replanted patients and their clinical significance

Objective To explore the dynamic changes of von willebrand factor(VWF)and Pseleetin in the finger-replanted patients,and the relationship between the prognosis of the surgery and hypercoagulability.Methods From December 2004 to December 2006,eishty finger-replanted patients were recruited to our study.with 40 healthy volunteers as controls.Plasma VWF and P-selectin were detected by enzyme-linked immunosorbent assay(EUSA)in both controls and patients before or after replantation.Results The VWF and P-selectin levels had significant differences between the replantations and controls(F=14.76,11.76,P＜0.01).The VWF levels in the patients of 1,4,8,16 hours after replantation were(1 715±493),(1 396±549),(1 266±504),(1 163±436)U/L respectively,all markedly higher than the controls(P＜0.01).The P-selectin levels in patients of 1,4,8,16,24 hours after operation were(14.7±2.6),(12.5±3.0),(11.8±3.2),(11.1±3.0)、(10.5±2.6)μg/L,significanfly higher than the controls(P＜0.01).The VWF levels in patients of pre-replantion and the 1,4,8,16,24,48,72 hours after replantation were(854±209),(1 535±389),(1 177±407),(1 040±283),(958±216),(829±193),(777±151),(713±137)U/L in successful group,and were(1 202±164),(2 333±243),(2 146±161),(2 039±244),(1 865±170),(1 645±283),(1 427±331),(1 188±262)U/L in unsuccessful groups.They were all significantly different at the same test-time points between two groups(t=4.44,5.12,6.10,8.43,10.17,8.85,5.10.4.61,P＜0.05).The P-selectin levels in patients of 1,4,8,16,24,48,72 hours after replantation were(13.9±2.5),(11.2±2.0),(10.2±1.6),(9.6±1.2),(9.2±0.9),(9.5±0.6),(9.3±0.4)μg/L in successful group,and(17.2±1.0),(16.9±1.0),(17.0±1.3),(16.1±1.1),(14.9±1.5),(13.8±1.4),(12.8±1.2)μg/L in unsuccessful group.Significant difference existed at the same testtime points between two groups again(t=5.22.9.91,10.35,12.79,9.46.9.45,9.33,P＜0.01).After replantation,both VWF and P-selectin were rapidly elevated and went to the summit 4 hours later,then declined to pre-replantation level about 24 to 48 hours later after replantation.Conclusions VWF and P-selectin were associated with the hypercoagulability.Dynamic monitoring VWF and p-selectin may be useful in determining the existence of hypercoagulability and the therapy of anti-coagulability. Key words: Finger injuries; Replantation; von Willebrand factor; P-Selectin

Epac Is Involved in cAMP-Mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells through the Activation of the Small GTPase Rap1.

Vascular endothelial cells provide a dynamic interface between circulating blood and underlying tissues that is critically involved in maintaining vascular integrity and homeostasis. The endothelium provides a surface for adhesion and subsequent extravasation of leukocytes to sites of inflammation. In addition, vascular endothelial cells are involved in the regulation of vascular tone, contribute to neo-vascularisation and mediate the formation of a platelet plug in the event of vascular damage. Rapid recruitment of bio-active components from intracellular storage pools has been shown to contribute to the critical role of endothelial cells in maintaining vascular homeostasis. A significant number of haemostatic components and inflammatory mediators originate from endothelial cell-specific, cigar-shaped organelles called Weibel-Palade bodies (WPBs) that function as storage vesicles for von Willebrand factor (VWF), a multimeric adhesive glycoprotein crucial for platelet plug formation, the leukocyte receptor P-selectin and a number of bioactive compounds that include the chemoattractants IL-8 and eotaxin-3. Following stimulation with agonists that increase intracellular Ca2+ levels, such as thrombin or histamine, WPBs fuse with the plasma membrane, thereby releasing their content onto the cellular surface or into the circulation. More recently, agonists such as epinephrine and vasopressin that raise intracellular levels of cAMP have been shown to promote the release of WPBs. The physiological importance of this pathway is illustrated by the rise in VWF levels in patients with von Willebrand's disease and mild hemophilia A following administration of the vasopressin analogue desmopressin (DDAVP) or epinephrine. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another, PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the exchange protein activated by cAMP (Epac) and its substrate, the small GTPase Rap1. SiRNA-mediated knockdown of the cAMP-GEF Epac1 results in decreased epinephrine-induced WPB exocytosis. Epinephrine stimulation of endothelial cells leads to activation of the small GTPase Rap1 in a PKA-independent fashion. In addition, siRNA-mediated knockdown of Epac1 completely abolished epinephrine-induced activation of Rap1 indicating that activation of Rap1 in response to epinephrine proceeds via Epac1. We subsequently addressed whether activation of Rap1 is crucial for WPB release. Downregulation of Rap1 activation through expression of Rap1GAP effectively inhibits epinephrine-induced WPB exocytosis. Taken together, these data uncover a new pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity

Cirrhosis of the liver is frequently accompanied by complex alterations in the hemostatic system, resulting in a bleeding tendency. Although many hemostatic changes in liver disease promote bleeding, compensatory mechanisms also are found, including high levels of the platelet adhesive protein von Willebrand Factor (VWF). However, conflicting reports on the functional properties of VWF in cirrhosis have appeared in literature. We have measured a panel of VWF parameters in plasma from patients with cirrhosis of varying severity and causes. Furthermore, we assessed the contribution of VWF to platelet adhesion, by measuring the ability of plasma from patients with cirrhosis to support adhesion of normal or patient platelets under flow conditions. VWF antigen levels were strongly increased in patients with cirrhosis. In contrast, the relative collagen binding activity, as well as the relative ristocetin cofactor activity, was significantly lower in patients as compared with controls, indicating loss of function. Accordingly, patients had a reduced fraction of high-molecular-weight VWF multimers. Both strongly elevated and reduced activity and antigen levels of the VWF cleaving protease ADAMTS13 were found in individual patients. Adhesion of either normal or patient platelets to a collagen surface was substantially increased when these platelets were resuspended in plasma of patients with cirrhosis, as compared with control plasma. In conclusion, highly elevated levels of VWF in patients with cirrhosis contribute to the induction of primary hemostasis despite reduced functional properties of the molecule. This phenomenon might compensate for defects in platelet number and function in patients with cirrhosis.