Unraveling the mystery of von Willebrand factor.
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Animals↗
Biomedical subjects
J A Van Mourik
Publications and source records attributed to J A Van Mourik.
Van Creveld, pioneer of hemophilia care and coagulation research in the Netherlands: a personal account.
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Blood Coagulation↗
Intracellular accumulation of factor VIII induced by missense mutations Arg593-->Cys and Asn618-->Ser explains cross-reacting material-reduced haemophilia A.
Patients with cross-reacting material (CRM)-reduced haemophilia A exhibit reduced levels of factor VIII antigen. In this study, we determined the molecular basis of the genetic defect in the factor VIII gene induced by either the Arg593-->Cys or the Asn618-->Ser missense mutation, identified in two CRM-reduced haemophilia A patients. We introduced either the Arg593-->Cys or the Asn618-->Ser mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNAs in C127 cells. Reduced levels of factor VIII activity and factor VIII antigen in conditioned medium of transfected cells indicated that the secretion of both factor VIII variants was impaired. The ratio of factor VIII antigen present in cell extract to that in conditioned medium was 1.9 and 2.4 times higher for rFVIII-R593C and rFVIII-N618S, respectively, than for rFVIII. Metabolic labelling and immunoprecipitation of transfected cells revealed that rFVIII-R593C and rFVIII-N618S persisted somewhat longer inside the cell than factor rFVIII. Intracellular accumulation and subsequent degradation of factor VIII-R593C and factor VIII-N618S may explain the reduced levels of both factor VIII activity and antigen in plasma of mild haemophilia A patients with corresponding genetic defects.
Cross Reactions↗
Substitution of Arg527 and Arg531 in factor VIII associated with mild haemophilia A: characterization in terms of subunit interaction and cofactor function.
The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527.
Amino Acid Substitution↗
Kinetics of factor VIII light-chain cleavage by thrombin and factor Xa. A regulatory role of the factor VIII heavy-chain region Lys713-Arg740.
Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region Lys713-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3 +/- 0.3 x 10(6) M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713-1637)-factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713-1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713-1637)-factor VIII is clearly reduced. Furthermore, the secondorder rate constant (0.2 +/- 0.1 x 10(6) M-1 s-1) of des-(713-1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713-1637)-factor VIII light-chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des-(713-1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis.
Antibodies, Monoclonal↗
The pro-polypeptide of von Willebrand factor is required for the formation of a functional factor VIII-binding site on mature von Willebrand factor.
We have established that a recombinant von Willebrand Factor (vWF) mutant (vWFdelpro) that lacks the propolypeptide, in contrast with mature wild-type vWF, with which it is identical in terms of primary amino acid sequence, is not able to form a complex with Factor VIII. Wild-type vWF (flvWF) and vWFdelpro were expressed in AtT-20 cells. Under the culture conditions employed, completely processed multimerized flvWF and dimeric vWFdelpro were secreted into the medium. FlvWF and vWFdelpro were compared for their Factor VIII-binding properties in two distinct assay systems. In a direct binding assay, purified human Factor VIII was shown to bind to flvWF that had been immobilized on the surface of microtitre wells by using an anti-vWF monoclonal antibody. In contrast, Factor VIII did not bind to immobilized vWFdelpro. In a competition assay, fluid-phase flvWF appeared to inhibit efficiently the binding of Factor VIII to immobilized vWF isolated from plasma, whereas vWFdelpro did not influence Factor VIII binding. From these observations, it is argued that the pro-polypeptide serves an essential role in the post-translational processes that lead to the expression of a functional Factor VIII-binding site on the mature vWF subunit.
Animals↗
Characterization of the enhanced adhesion of neutrophil leukocytes to thrombin-stimulated endothelial cells.
We have characterized the mechanisms by which thrombin enhances neutrophil leukocyte (PMN) adhesion to human endothelial cells in vitro. Thrombin rapidly and transiently increased PMN adhesion by an action on the endothelial cells. The transience of the response was due to at least two factors: desensitization of the endothelial cell responsiveness to thrombin in the continued presence of the agonist; and the lability (t1/2 less than 15 min) of the effector molecules expressed by the endothelium. Experiments with exogenous platelet-activating factor (PAF) and with PAF antagonists demonstrated that PAF production, although it may facilitate the enhanced PMN adhesion seen in response to thrombin, is not sufficient to explain the reaction. By using a variety of antibodies directed against cell surface ligands, and comparing adhesion of PMN to endothelium and to protein-coated surfaces, we deduce that several endothelial ligands not previously reported as playing a role in PMN adhesion are involved in these interactions. Of particular interest was the finding that antibodies recognizing two thrombin-regulated endothelial cell surface ligands, GMP-140 and the CD63-related Ag, both inhibited adhesion of PMN to thrombin- or LPS-pretreated endothelium. We conclude that thrombin acts to enhance PMN adhesion to endothelium at least in part by transiently altering the conformation or level of expression of these ligands.
Antigens, CD↗
A three-dimensional model system to study the interactions between human leukocytes and endothelial cells.
Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.
Antibodies, Monoclonal↗
Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments.
The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3'-ends by selective restriction-enzyme digestion and used as templates for 'run-off' mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.
Antibodies, Monoclonal↗
The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII.
The interaction between human Factor VIII and immobilized multimeric von Willebrand Factor (vWF) was characterized. Equilibrium binding studies indicated the presence of multiple classes of Factor VIII-binding sites on vWF. The high-affinity binding (Kd = 2.1 x 10(-10) M) was restricted to only 1-2% of the vWF subunits. Competition studies with monoclonal antibodies with known epitopes demonstrated that the Factor VIII sequence Lys1673-Arg1689 is involved in the high-affinity interaction with vWF.
Amino Acid Sequence↗
Factor VIII binds to von Willebrand factor via its Mr-80,000 light chain.
The formation of a complex between factor VIII (FVIII) and von Willebrand factor (vWF) was studied using purified radiolabeled human FVIII and purified human vWF. A binding assay was developed in which vWF was coated on microtiter wells. FVIII was shown to bind specifically and reversibly to the immobilized vWF. At a coating of 70 pg vWF/well, binding was half-maximal at a FVIII concentration of 70 +/- 10 pM. In order to ascertain which part of FVIII interacted with vWF, eight monoclonal antibodies, directed against FVIII, were tested for their ability to inhibit FVIII-vWF interaction. One of the eight antibodies, CLB-CAg:58, inhibited binding completely. This antibody was demonstrated to react with the Mr-80,000 light chain of FVIII. Direct evidence for the involvement of this chain in vWF binding was obtained by studying the binding of isolated, radiolabeled FVIII heavy and light chains. In a typical experiment 23-30% of the radioactivity bound when the FVIII light chain was added and less than 1% when the FVIII heavy chain was added.
Antibodies, Monoclonal↗
The role of thrombin in the regulation of the endothelial prostaglandin production.
Prostaglandin synthesis in endothelial cells may be initiated by the addition of exogenous substrate (arachidonic acid) or by addition of thrombin or the CA2+-ionophore A23187, which leads to prostacyclin formation from endogenous substrates. We noticed that endothelial cells produce more than twice the amount of prostacyclin when incubated with thrombin and arachidonic acid together than with arachidonic acid alone. In addition, it was found that the thrombin-induced conversion of endogenous substrates was inhibited by exogenous arachidonic acid. This means that the conversion of exogenous added arachidonic acid to prostacyclin was stimulated by thrombin. This activation of the enzymes involved in prostacyclin synthesis lasted about 5 min and could be inhibited by phospholipase inhibitors such as mepacrine and p-bromophenyl-acylbromide but not by the cAMP analogue dibutyryl cAMP, an inhibitor of arachidonic acid release from cellular phospholipids. These data demonstrate that, in addition to causing release of endogenous substrate, thrombin and the Ca2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid.
Arachidonic Acid↗
Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells.
Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca2+ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160-168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g X ml-1), and a dense one with a peak density of 1.12 g X ml-1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca2+ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.
Blood Coagulation Factors↗
The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells.
Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca2+-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca2+ but independent of intracellular cAMP.
Antigens↗
Arachidonate metabolism in cultured human vascular endothelial cells. Evidence for two prostaglandin synthetic pathways sensitive to acetylsalicylic acid.
The effect of acetylsalicylic acid on endothelial prostaglandin synthesis was measured in the presence of exogenous and endogenous substrates. In both types of measurement, a rate of inhibition was found similar to that observed for acetylsalicylic acid inhibition of cyclooxygenase activity in platelets. After withdrawal of acetylsalicylic acid, a rapid restoration of cyclooxygenase activity was observed when exogenous [1-14C]arachidonate was used as a substrate (50% of initial activity at 5 h). However, when endogenous substrate, released after phospholipase activation induced by thrombin treatment of the cells, was used to test cyclooxygenase activity, only partial restoration of enzymic activity was observed (30% after 48 h). Phospholipase activity, measured by the release of free fatty acids, was not inhibited by acetylsalicylic acid. Measurement of turnover times by incubating the cells with cycloheximide revealed a short turnover time for the enzymic activity tested with exogenous [1-14C]arachidonate (2.3 h) and a relatively long turnover time for the cyclooxygenase activity tested with endogenous substrate released after thrombin treatment of the cells (54 h). These results suggest that at least two pools of cycloxygenase are involved in endothelial prostaglandin synthesis.
6-Ketoprostaglandin F1 alpha↗
Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells.
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Cells, Cultured↗
Bradykinin-mediated hypotension after infusion of plasma-protein fraction.
In patients who required volume expansion during extracorporeal circulation, the plasma bradykinin concentration was monitored simultaneously with the mean arterial pressure during infusion of either albumin solution or PPF. The PKA content of the PPF and the albumin solution was 29 and 3 U/L, respectively, measured spectrophotometrically. In six patients receiving 250 ml of PPF, the mean arterial pressure decreased 22% to 54% within 1.5 min after infusion, whereas the plasma bradykinin concentration, measured by radioimmunoassay, increased significantly (p less than 0.0005) during the first minute. In six patients receiving 250 ml of 4% albumin solution, no blood pressure changes were found, and the plasma bradykinin concentration rose only slightly. In vitro, linear correlation (r = 0.94, p less than 0.0005) was observed between the level of PKA of 26 different lots of PPF and the concentrations of bradykinin that were generated in Hageman factor-deficient plasma after incubation with PPF. It is concluded that the hypotensive reactions observed after PPF infusion during extracorporeal circulation are caused by the PKA-induced bradykinin generation.
Adult↗
Hypotensive effects of plasma protein fraction. The relation between prekallikrein activator, bradykinin generation, and blood pressure in an animal model.
In a rat model, the relationship between the activity of PKA in human PPFs, the changes in arterial BK concentration, and the changes in blood pressure on infusion of PPF was investigated. The rat was chosen as a model because it is reported to be one of the few animals sensitive to human PKA. However, hypotensive reactions after infusion of human PKA-containing PPF were observed only in the presence of a bradykinin-potentiating peptide (BPP9a). A correlation was found between the PKA content of the rapidly infused PPF, the BK generation in the rat, and the fall in arterial blood pressure. In control experiments, infusions of BK provoKed a similar fall in blood pressure at corresponding BK levels. After neutralization of the PKA activity in the PPF with C1-esterase inhibitor, neither a rise in BK level nor a fall in blood pressure was observed on infusion. We conclude therefore that the hypotensive reactions were caused by PKA-mediated generation of BK.
Animals↗