Platelet von Willebrand factor: Comparison with plasma von Willebrand factor

Platelet von Willebrand factor (vWf) was compared to its plasma counterpart. The platelet vWf was different from plasma vWf in that 1) the multimeric organization was different, 2) larger multimers were present, and 3) the ratio of vWf activity to antigen was higher. Platelet and plasma vWf were similar in their antigenic reactivity in the electroimmunoassay and by liquid phase radioimmunoassay. The amount of vWf activity in large platelets was significantly higher than in normal platelets while the antigen content, although somewhat higher, was not significant. These studies show differences between normal platelet and plasma vWf, and also suggest that platelet size must be considered when platelet vWf is measured in disease states.     

Analysis of serum soluble CD40 ligand in patients with influenza virus-associated encephalopathy

CD40 ligand (CD40L) is mainly expressed on activated platelets and CD4+T cells, and it can be cleaved from the cell surface, releasing a soluble CD40L (sCD40L). Most sCD40L is derived from activated platelets. A previous paper revealed that the platelet number of patients with influenza virus-associated encephalopathy (IE) was correlated with the outcome. We determined the utility of sCD40L as a predictor for the prognosis of IE. We measured the serum concentration of sCD40L and the platelet number on the day of hospitalization in 34 patients with IE, 16 with influenza virus-associated febrile seizures (IFS), 19 with influenza virus infection without complications (Flu), and 7 with Epstein-Barr virus (EBV) infection. The serum sCD40L concentrations in IE and IFS were significantly lower than those in controls, Flu, and EBV infections. Serum sCD40L concentrations in the IE group were 0.70+/-0.43 ng/ml for deceased patients, 1.73+/-1.36 ng/ml for those with sequelae, and 3.85+/-2.91 ng/ml for those without sequelae. There was no significant difference in platelet number between IE patients with and without sequelae, while the platelet number of deceased patients with IE was significantly lower than in controls, Flu, and IFS. Serum sCD40L concentration on the day of hospitalization was more correlated with the outcome of IE than platelet number. Our findings suggest that the serum sCD40L concentration during acute IE is important for predicting the prognosis at an early stage.     

Diurnal variation of soluble CD40 ligand in patients with acute coronary syndrome. Soluble CD40 ligand and diurnal variation

Introduction

We sought to investigate whether day-night variations occur in the concentration of circulating soluble CD40 ligand in patients with acute coronary syndrome, as this may have practical implications.

Materials and Methods

We assessed 70 consecutive ST-segment elevation myocardial infarction patients admitted into the Coronary Care Unit and 50 control subjects. Each subject was studied under strictly controlled light/dark conditions. Blood samples were drawn at 09:00 h (light phase) and 02:00 h (dark phase). Nocturnal blood samples were drawn by a trained nurse, with the help of a minute torch with a dim red light in order to avoid any direct lighting on the patient during sleep. The soluble CD40 ligand was measured using a commercially available ELISA.

Results

Soluble CD40 ligand levels showed no diurnal variations in control subjects. In the ST-segment elevation myocardial infarction group, however, soluble CD40 ligand concentration (pg/mL) in the light phase was significantly higher than that in the dark phase (167.3 ± 63.2 vs 118.9 ± 48.3 pg/mL, p < 0.001).

Conclusions

The study shows for the first time the existence of diurnal variations in soluble CD40 ligand levels in ST-segment elevation myocardial infarction patients, which indicates the need for standardizing the time of blood sampling for the assessment of this molecule, at least in studies involving ST-segment elevation myocardial infarction patients.     

Release of soluble CD40 ligand after platelet activation: studies on the solubilization phase

sCD40L is released from platelets as a soluble, proteolyzed form of CD40 ligand (CD40L; CD154) which is exposed on the surface after platelet activation. Ethylenediaminetetraacetate (EDTA), the CD40-blocking antibody G28-5, and GPIIb-IIIa antagonists are known to inhibit the solubilization when added prior to activation. It is assumed that the surface expression of CD40L is a result of a separate fast process and that the solubilization is secondary to this. The release of sCD40L in this solubilization phase has been studied; that is, inhibitory substances were added to platelet-rich plasma (PRP) 10 min after addition of the activation agonist (100 microM SFLLRN), at which time the secretion phase was over as tested with beta-thromboglobulin (beta-TG). G28-5 (10 microg/ml) and EDTA (5 mM) inhibited the solubilization phase which did not require the presence of an activation agonist. Prostaglandin E1 (PGE1; 20 microM) and cytochalasin D (C8273; 60 and 100 microM), which exert their effects intracellularly, inhibited the solubilization even in the presence of abciximab (ReoPro; 40 microg/ml). The intracellular effect was not related to CD40L-containing microparticles as demonstrated by ultracentrifugation. Intracellular alkalinization by preincubation of PRP with 20 mM NH4Cl for 60 min resulted in a small but reproducible reduction in the amount of extracellular sCD40L. SFLLRN induced solubilization of CD40L also from the platelets of a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa, albeit at a lower rate than from normal platelets, and fibrinogen enhanced the solubilization from washed normal platelets. The data show that the solubilization of CD40L not only depends on reactions on the platelet surface but also that intracellular structures are engaged even during the solubilization phase.     

Validation of a rapid test (VWF-LIA) for the quantitative determination of von Willebrand factor antigen in type 1 von Willebrand disease diagnosis within the European multicenter study MCMDM-1VWD

Background

Accurate measurement of von Willebrand factor (VWF) is a critical requirement for the diagnosis of von Willebrand disease (VWD).

Aim of the study

To evaluate the diagnostic efficiency of a rapid quantitative test for the measurement of VWF antigen (VWF:Ag) in type 1 VWD.

Patients and methods

VWF:Ag was measured with an ELISA in a robotic instrument, as a reference method, and with a fully automated latex-immunoassay (LIA) on an ACL 9000 analyser in 1,716 subjects enrolled within the Molecular and Clinical Markers for Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) Study. Among these subjects, 1,049 were healthy controls, 281 healthy family members and 386 affected members from 127 European families with type 1 VWD.

Results

The assay linearity range was 10-125 IU/dL for LIA (R² = 0.99) and 5-133 IU/dL for ELISA (R² = 0.99). The inter-assay CV for low VWF levels (∼ 30 IU/dL) was 2% for the LIA test and 8.7 % for ELISA. The sensitivity for detection of type 1 VWD affected members was 86% and the specificity 91% for LIA, 87% and 90% for ELISA. A receiver-operator (ROC) analysis disclosed only a marginal difference between the two tests, LIA having a slightly greater area under the curve (0.94 vs. 0.93, p = 0.03).

Conclusion

VWF:Ag LIA compared well to standard ELISA in this large population of patients and controls, showing better CV. However the lower detection limit for the VWF:Ag LIA compared to the VWF:Ag ELISA means that the LIA assay is less good at discriminating between type 3 VWD and moderate type 1 VWD.     

Allelic frequencies of three VNTRs in intron 40 of the human von Willebrand factor gene in types 1, 2, and 3 von Willebrand disease patients and controls of a Brazilian population

Intron 40 of the human von Willebrand factor (vWF) gene contains a polymorphic region with three variable-number tandem repeats (VNTRs), type (ATCT)_n. In the present report, we evaluated the allelic frequencies of these three VNTRs in a population constituted by 51 Brazilian Caucasian and 25 Types 1, 2, and 3 von Willebrand disease (vWD) patients, and performed segregation analysis in eight families affected by vWD Types 1 and 2. Three pairs of primers were used to amplify independently nucleotides 1640–1794 (VNTR 3), 1890–1991 (VNTR 1), and 2215–2396 (VNTR 2) from intron 40. The observed heterozygosities (0.86, 0.66, and 0.66 for VNTRs 3, 1, and 2, respectively) were in accordance with the expected heterozygosities derived from the allele frequencies (0.81, 0.64, and 0.70, respectively). Although the three VNTRs were highly polymorphic, VNTR 3 showed the highest values of heterozygosity [Haemostasis 25 (1995) 264; Hum. Mol. Genet. 1 (1992) 287.].     

Modulation of the von Willebrand factor-dependent platelet adhesion through alternative proteolytic pathways

Introduction

Platelet adhesion to collagen under high shear rates depends on the optimal size of the von Willebrand factor (VWF) multimers, which is determined by their limited proteolysis. The present study attempts to identify the role of hemostatic-fibrinolytic enzymes (thrombin, plasmin) and leukocyte-derived proteases (matrix metalloproteinase (MMP)-8, MMP-9, neutrophil elastase) in the cleavage of VWF and to characterize the effect of flow and platelets on this proteolysis and its functional consequences on platelet adhesion.Methods and resultsAccording to VWF immunoblots, plasmin, neutrophil elastase and thrombin at concentrations of in vivo relevance resulted in extensive degradation of VWF within several minutes. Platelets protected VWF against this proteolysis under static conditions, whereas perfusion of the proteases at 3350 s^-1 shear rate over VWF immobilized on artery cross sections enhanced its degradation and blocked the protective effect of platelets. In parallel with VWF digestion, the examined proteases impaired the VWF-dependent platelet adhesion as reflected in the decreased surface-bound GpIIb/IIIa immunoreactivity following perfusion of collagen-coated surfaces or artery sections with blood and plasmin, neutrophil elastase or thrombin. Within the time frame of minutes no VWF cleavage could be detected under static or flow conditions after exposure to MMP-8 and MMP-9 at concentrations relevant to physiological neutrophil counts.

Conclusion

Our results indicate a shear- and platelet-dependent role for several proteases in the local modulation of the VWF function.     

Effects of alimentary lipemia and inflammation on platelet CD40-ligand

INTRODUCTION: In patients with chronic hypercholesterolemia, the CD40-CD40L dyad is upregulated, contributing to the initiation and progression of atherosclerosis. Our aim was to describe the role of postprandial lipemia and inflammatory stimulation on platelet and monocyte activation and CD40-ligand (CD40L) levels. METHODS AND RESULTS: Before and 2 h after consumption of a defined fatty meal, whole blood samples of 31 healthy subjects were incubated with endotoxin (LPS). CD40-ligand and CD62P expression on platelets, tissue-factor expression on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Soluble CD40-ligand plasma levels were measured with an ELISA. After the meal, serum triglyceride levels increased from 137.6+/-60.5 mg/dl to 201.5+/-75.0 mg/dl. Expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. No significant changes after the meal were observed concerning tissue factor expression on monocytes and platelet-monocyte aggregates. Addition of LPS showed no significant effect concerning CD40L or CD62P expression on platelets, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal. CONCLUSIONS: Acute alimenatry lipemia leads to a decreased expression of CD40L on platelets and a reduced plasma level of sCD40L, suggesting an increased turnover in the CD40L system. CONDENSED ABSTRACT: Before and after a fatty meal, blood samples of 31 healthy subjects were incubated with LPS. After the meal, expression of CD40L and CD62P on platelets and plasma levels of soluble CD40L were significantly decreased. Addition of LPS showed no effect concerning CD40L or CD62P expression, whereas the amount of platelet-monocyte aggregates significantly increased under LPS stimulation after the fatty meal.     

CD40 ligand as a potential biomarker for atherosclerotic instability

Abstract

Objective: The signaling protein CD40 and its ligand, CD40L, are thought to contribute to atherosclerotic plaque formation and rupture. We sought to determine their utility as markers of cerebral atherosclerosis and neurological dysfunction.

Methods: We recruited 82 patients with acute cerebral infarction (ACI) and classified each as having large-artery atherosclerosis (LAA, 30), small-artery occlusion (36), or cardioaortic embolism (16). We also recruited 17 patients who had carotid artery stenosis (CAS) without stroke and 20 healthy individuals as controls. CD40L expression on peripheral blood monocytes (PBMCs) was detected by direct immunofluorescence with flow cytometry, and serum soluble CD40L (sCD40L) was measured by ELISA.

Results: CD40L expression on PBMCs was highest in the LAA group, followed by that in the CAS group (both P < 0·01 versus control). It was also higher in patients with atherosclerotic infarction than in those without atherosclerosis (P < 0·05). PBMC CD40L expression was sensitive and specific for detecting atherosclerosis and LAA cerebral infarction and was superior to serum C-reactive protein for predicting cerebral atherosclerosis (P < 0·01). Serum sCD40L was higher in patients with ACI than in healthy controls (P < 0·01) or patients with CAS (P < 0·01) and correlated with increased disability on three scales (all P < 0·01).

Conclusions: We conclude that patients with ACI have an upregulated CD40–CD40L system, which could be used as a clinical biomarker for assessing atherosclerotic instability and severity of neurological dysfunction.     

Multimeric analysis of von Willebrand factor in megakaryocytes

von Willebrand factor (vWF) is a glycoprotein that appears to play a major role in subserving the adhesion of platelets to subendothelium during hemostasis. Endothelial cells have been shown capable of synthesizing and releasing this large, multimeric glycoprotein that normally circulates in the plasma in association with the factor VIII coagulant molecule. Megakaryocytes, the precursor cells of blood platelets, also appear to possess vWF biosynthetic capacity, since cultured guinea pig megakaryocytes have been shown to produce a polypeptide precipitable by antibody to vWF. We now report a study of the multimeric structure of vWF in the megakaryocyte, as well as a quantitative comparison of megakaryocyte vWF with that of platelets and plasma in the guinea pig. Multimeric analysis on SDS agarose gels employing ¹²⁵I-emu anti-human vWF revealed striking homology between human and guinea pig vWF. Platelets and megakaryocytes from the same guinea pigs contained vWF of highly comparable multimeric composition. Moreover, megakaryocytes and platelets both contained a subset of very high molecular weight multimers not present in plasma. Quantitation of vWF in megakaryocytes and platelets was achieved with a radioimmunoassay performed on detergent (NP-40) lysates of washed cells. These measurements showed that megakaryocytes and platelets contain 0.079 and 0.069 U of vWF per mg protein, respectively. The results of these studies suggest that megakaryocytes represent the primary, if in fact not sole, source of platelet vWF.     

Increased levels of plasma von Willebrand factor in migraine crisis

Introduction – To evaluate the participation of the vessel wall in the pathogenesis of migraine attack, we measured the plasma levels of von Willebrand factor (vWF), a protein secreted from the endothelial cells. Material & methods – 17 patients suffering from migraine without aura and 25 healthy volunteers were studied. von Willebrand factor and platelet aggregation tests were studied by conventional methods. Results – The levels of vWF:antigen increased from 72.4 ± 29 U/dl in the intercrisis to 130.2 ± 75 U/dl during the attack (p < 0.01). We did not detect difference in the platelet aggregability in both phases. Plasma vWF activity measured as ristocetin cofactor (vWF:RCo) was similar in intercrisis and crisis (100.6 ± 31 U/dl vs 94.5 ± 44 U/dl). Conclusions – There is a plasma release of vWF molecules during the migraine crisis. This feature is not platelet dependent and is probably a consequence of endothelial stress.     

Deficiency of von Willebrand factor-cleaving protease activity in the plasma of malignant patients

von Willebrand factor (vWF) multimeric pattern and von Willebrand factor-cleaving protease activity (vWF-cp) were studied using plasmas from patients with advanced stage- and limited stage-malignant tumors. Deficiency of highly polymeric forms of vWF was observed in plasmas from 7 of 11 patients tested. vWF-cp activity was deficient in plasma samples of six patients with advanced stage-malignant tumors (ranging from 6% to 30% activity of normal plasma), whereas an essentially normal vWF-cp activity was observed in samples taken from patients with limited stage-malignant tumors. Inhibitor of vWF-cp was not detected in any plasma samples tested. To further analyze the relevance of this enzymatic activity in metastatic diagnosis, a study of vWF-cp activity was conducted in 17 patients with colon cancer, and it was shown that deficiency of vWF-cp was associated with the progression of the disease.     

Unbalanced expression of ADAMTS13 and von Willebrand factor in mouse endotoxinemia

INTRODUCTION: Secondary ADAMTS13 deficiency may occur in septic patients. The expression of ADAMTS13 in mouse endotoxinemia was studied. METHODS: The blood and mRNA expression levels of ADAMTS13 and von Willebrand factor were measured in lipopolysaccharide-injected mice. RESULTS: The plasma ADAMTS13 activity in wild-type mice was significantly decreased at 2 h after lipopolysaccharide injection, and this decrease in ADAMTS13 activity preceded the decrease in ADAMTS13 mRNA expression in the liver and continued for 24 h. However, no decreases in the plasma ADAMTS13 activity after lipopolysaccharide injection were observed in mice pretreated with a neutrophil elastase inhibitor or in plasminogen-deficient mice, suggesting that the decrease in ADAMTS13 activity was processed efficiently by the coordinated actions of plasmin and neutrophil elastase. von Willebrand factor mRNA was abundantly expressed in the lung and moderately in the kidney, but showed relatively low expression in the liver without lipopolysaccharide injection. However, von Willebrand factor mRNA expression in the liver was significantly increased after lipopolysaccharide injection and this high expression level continued for 24 h after the injection. The von Willebrand factor and ADAMTS13 mRNA expression levels in these organs changed in the opposite manners following lipopolysaccharide administration. Furthermore, the blood von Willebrand factor level increased after lipopolysaccharide administration, in contrast to the decrease in the blood ADMTS13 level after lipopolysaccharide administration. CONCLUSION: These data suggest that imbalance between the blood von Willebrand factor and ADAMTS13 levels may occur in endotoxinemia, and that this may partly contribute to the thrombotic state associated with endotoxinemia.     

Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

Introduction

VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions.

Methods

The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy.

Results

The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration.

Conclusions

The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.     

Plasma von Willebrand Factor and Intestinal Ischaemia-Reperfusion Injury in Rats

The purpose of this study was to determine whether plasma von Willebrand factor concentrations are correlated with the degree of intestinal ischaemia-reperfusion injury. Forty-six anaesthetised adult Wistar rats were divided into five groups. The sham-operated group (S, n=10) had laparotomy and isolation of the superior mesenteric artery without clamping. Three ischaemia-reperfusion groups (n=10 in each) had clamping of the superior mesenteric artery for 15, 30, and 45 minutes, respectively, and reperfusion for 15 minutes. A control group (C, n=6) had direct puncture of the heart to sample blood. Mean arterial pressure was measured continuously. Blood was collected at the end of the study to measure von Willebrand factor. The small bowel injury was graded histologically. There was a significant systemic hypotension after declamping in all ischaemia-reperfusion groups, which had a high negative correlation with the histological score (R=−0.46, F=10.1, p<0.003, simple linear regression). Plasma von Willebrand factor was significantly elevated in the three ischaemia-reperfusion groups compared with the control group but not significantly different from the sham-operated group (mean von Willebrand factor concentration (SEM): 156 (29), 283 (29), 295 (25), 381 (44), and 366 (40)% in C, S, ischaemia-reperfusion 15, ischaemia-reperfusion 30, and ischaemia-reperfusion 45 groups, respectively). The concentration of von Willebrand factor was not correlated to the histological score (R=0.22, F=1.83, p<0.2) or the degree of hypotension after the removal of the clamp (R=−0.22, F=1.8, p<0.2, simple linear regression). This study shows that von Willebrand factor concentration does not correlate with the degree of intestinal ischaemia-reperfusion injury. It is unlikely that von Willebrand factor can be used as a predictor of disease severity.     

Binding of porcine von Willebrand factor to human platelets in the presence of ristocetin

The interaction of porcine von Willebrand factor (vWF) with human platelets in the presence of ristocetin was examined. Binding was rapid, specific, saturable and dependent on ristocetin concentration with half-maximal stimulation occuring at a ristocetin concentration of 0.5mM. Assuming vWF to be a tetramer with a MW of 9.5×10^?5, approximately 94,000 vWF binding sites per platelet were found with an average binding constant of 2.1×10^?8M. Humam vWF competed with the porcine protein for this site only in the presence of ristocetin. Binding of porcine vWF to platelets was found to be less sensitive to reduction with dithiothreitol than platelet agglutinating activity.