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The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein–coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca2+- or cAMP-raising agonists.

The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein–coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca2+- or cAMP-raising agonists.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

Before de novo synthesized von Willebrand factor (vWF) leaves the endothelial cell, it undergoes endoproteolytic cleavage of its propeptide (vW antigen II). The processed vWF and propeptide are either released constitutively or, following activation of the endothelium, released through the regulated pathway. In a recent study (Borchiellini et al, Blood 88:2951, 1996), we showed that the half-life of mature vWF and of its propeptide differ fourfold to fivefold. We postulated that the molar ratio of the propeptide to mature vWF could serve as a tool to assess the extent of endothelial cell activation under physiologic and clinical conditions. To test this hypothesis, we measured mature vWF and propeptide in patients with documented acute and chronic vascular disease, including patients with thrombotic thrombocytopenic purpura (TTP), acute septicemia, and diabetes mellitus. These data were compared with experimental conditions in healthy subjects in which perturbation of the endothelium was simulated by physical exercise or by administration of 1-deamino-8-D-arginine vasopressin (DDAVP) or endotoxin. In all individuals of the latter study group, both vWF and propeptide levels were elevated during the acute phase of the experimentally induced vascular perturbation; at later time points after stimulation, only vWF levels remained elevated. In patients with sepsis and TTP, both vWF and propeptide were elevated several-fold. Thus, this pattern can readily be explained in terms of acute perturbation of the endothelium. In contrast, in patients with diabetes mellitus propeptide levels were only slightly elevated, whereas vWF levels were elevated twofold to threefold. This pattern is a typical feature of chronic, low-grade activation of the endothelium. These observations support our hypothesis that measurement of both propeptide and vWF levels allows to discriminate between chronic and acute phases of endothelial cell activation in vivo. Measurement of only vWF is less indicative in this respect.

Before de novo synthesized von Willebrand factor (vWF) leaves the endothelial cell, it undergoes endoproteolytic cleavage of its propeptide (vW antigen II). The processed vWF and propeptide are either released constitutively or, following activation of the endothelium, released through the regulated pathway. In a recent study (Borchiellini et al, Blood88:2951, 1996), we showed that the half-life of mature vWF and of its propeptide differ fourfold to fivefold. We postulated that the molar ratio of the propeptide to mature vWF could serve as a tool to assess the extent of endothelial cell activation under physiologic and clinical conditions. To test this hypothesis, we measured mature vWF and propeptide in patients with documented acute and chronic vascular disease, including patients with thrombotic thrombocytopenic purpura (TTP), acute septicemia, and diabetes mellitus. These data were compared with experimental conditions in healthy subjects in which perturbation of the endothelium was simulated by physical exercise or by administration of 1-deamino-8-D-arginine vasopressin (DDAVP) or endotoxin. In all individuals of the latter study group, both vWF and propeptide levels were elevated during the acute phase of the experimentally induced vascular perturbation; at later time points after stimulation, only vWF levels remained elevated. In patients with sepsis and TTP, both vWF and propeptide were elevated several-fold. Thus, this pattern can readily be explained in terms of acute perturbation of the endothelium. In contrast, in patients with diabetes mellitus propeptide levels were only slightly elevated, whereas vWF levels were elevated twofold to threefold. This pattern is a typical feature of chronic, low-grade activation of the endothelium. These observations support our hypothesis that measurement of both propeptide and vWF levels allows to discriminate between chronic and acute phases of endothelial cell activation in vivo. Measurement of only vWF is less indicative in this respect.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

A monoclonal antibody directed against the von Willebrand factor moiety (vWF) of factor Vlll-von Willebrand factor (FVIII-vWF), which blocks ristocetin-induced platelet aggregation as well as the binding of FVIII-vWF to platelets in the presence of ristocetin, inhibited platelet adherence to human artery subendothelium when present in normal flowing blood. This monoclonal antibody, CLB-RAg 35, inhibited platelet adherence as a function of the shear rate. At wall shear rates below 500 s-1, platelet adherence was not affected, but at higher shear rates platelet adherence was gradually inhibited, reaching an average of 11% of the normal value at 2,500 s-1. Indirect immunofluorescence established the reactivity of CLB-RAg 35 with vWF present in artery subendothelium. Pretreatment of normal vessel walls with this antibody inhibited adherence of platelets in blood from a patient with severe homozygous von Willebrand's disease and in blood from normal individuals. The inhibition was shear-rate dependent and significant at high shear rates (2,500 s-1). By adding increasing amounts of purified FVIII-vWF to normal blood, the inhibition was gradually overcome. These data indicate that vWF present in the vessel wall contributes appreciably to platelet adherence. At high wall shear rates, platelet adherence is mediated virtually completely by both plasma FVIII-vWF and vWF in the vessel wall. At low wall shear rates (below 500 s-1), platelet adherence occurs independent of FVIII-vWF in plasma and vWF in the vessel wall.

We have studied the role of factor VIII-von Willebrand factor (FVIII- vWF) in both platelet adherence to subendothelium and ristocetin- induced platelet aggregation using monoclonal antibodies to human FVIII- vWF. Twenty-five monoclonal antibodies were obtained, two of which were directed to the factor VIII moiety of FVIII-vWF; one of these two completely inhibited the procoagulant activity (FVIII:C). The remaining 23 monoclonal antibodies were directed to the von Willebrand factor moiety of FVIII-vWF. The ability of the latter monoclonal antibodies to inhibit platelet adherence to arterial subendothelium was investigated with a perfusion model. According to the number of platelets adhering to the subendothelium, three groups of monoclonal antibodies could be discerned: (A) antibodies not affecting platelet adherence; (B) antibodies that inhibited platelet adherence to the level as observed when von Willebrand's disease plasma was tested; and (C) antibodies that completely inhibited both platelet adherence to subendothelium and ristocetin-induced platelet aggregation. The two antibodies present in group C competed for the same or closely related epitope(s) present on FVIII-vWF. These results demonstrate that a domain is present on the FVIII-vWF molecule that is associated both with ristocetin-induced aggregation and with the ability of FVIII-vWF to support platelet adherence to the subendothelium. Based on these observations, it is concluded that ristocetin-induced binding of FVIII-vWF to platelets reflects, at least in part, a physiologic mechanism regulating the function of FVIII-vWF in primary hemostasis.

Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa–factor VIIIa complex assembly.

Endothelial cells synthesize a heterodimeric adhesion molecule, the vitronectin receptor (VnR), which is similar to the platelet glycoprotein (GP)IIb/IIIa complex. The subunits of the endothelial VnR (VnR alpha and GPIIIa) have been studied for their ability to express alloantigens associated with platelet GPIIb and IIIa. We previously showed that endothelial GPIIIa can express the platelet alloantigen Zwa or PIA1, which is associated with GPIIIa. We studied the relationship between the expression of Zwa on platelets and endothelial cells in neonates (n = 13). Using immunoprecipitation and immunofluorescence techniques, we showed that the Zwa antigen is either expressed or absent from both platelets and endothelial cells of the same individual. This finding indicates that in both cell types the same gene is expressed. We also showed that Zwa-negative endothelial cells express Zwb (PIA2), in analogy to Zwa-negative platelets. Moreover, our results strongly suggest expression on endothelial cells of Yukb, a recently described platelet alloantigen, also located on GPIIIa. However, we could not demonstrate expression on the endothelial VnR alpha subunit of Baka, an alloantigen located on platelet GPIIb. These findings are in agreement with the concept that the endothelial GPIIIa subunit is more closely related to its platelet counterpart than to the endothelial VnR alpha subunit.

Since dialysis of human factor VIII against buffers of low ionic strength yielded two distinct components, and since the factor VIII fraction isolated from normal plasma showed von Willebrand factor activity as defined by the corrective effect on abnormal platelet retention and ristocetin aggregation in von Willebrand’s disease, the present studies were performed to determine if the correcting activities could be attributed to one or both of the components. Dialysis of factor VIII against buffers of low ionic strength led, however, to a decrease in factor VIII procoagulant activity and the reduction of the correcting activities, which suggested that the intact aggregate was required for procoagulant activity and for von Willebrand factor activity. In this respect dialysis of factor VIII at low ionic strength differed from dissociation at high salt concentrations. The two low ionic strength components were identified by the use of a rabbit antiserum against factor VIII, and could be distinguished on the basis of specific antigenic structures. Dialysis of factor VIII at low ionic strength led to a decrease in antigenic determinants closely related to factor VIII function. Specific antibodies to the low ionic strength components inhibited factor VIII activity in normal plasma, but the residual factor VIII activity was higher than that after inhibition with antibodies against intact factor VIII. Both antibodies interfered with von Willebrand factor activity.

The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, is, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the effects of the interaction between human monocytes and endothelial cells on the production of type 1 plasminogen activator inhibitor (PAI-1) by endothelial cells. The effects of adherence and transendothelial migration of monocytes on endothelial PAI-1 release were compared with those of other leukocytes, conditioned media from monocytes, and interleukin-1β (IL-1β). Because the cell culture system used allows simultaneous analysis of the lumenal and the subendothelial compartment of endothelial cell monolayers, we also studied into which direction PAI-1 is released by endothelial cells. Under quiescent conditions, the net amount of PAI-1 accumulated at the lumenal side was twofold higher than that accumulated at the subendothelial side (about 2.0 μg PAI-1/106cells and 1.1 μg PAI-1/106cells, respectively, in 24 hours), as analyzed by a quantitative immunoradiometric assay (IRMA). Direct cell-cell contact between highly purified monocytes and endothelial cells strongly enhanced the PAI-1 release by endothelial cells in a dose-dependent way, whereas lymphocytes and neutrophils did not affect endothelial PAI-1 production. The monocyte-mediated increase was first detected after 12 hours of incubation and lasted for at least 48 hours. In the presence of two monocytes per endothelial cell, the increases of PAI-1 at the lumenal side and at the subendothelial side were 87% and 32% in 24 hours, respectively. The effect of IL-1β on PAI-1 release by endothelial cells closely resembled that observed for monocytes. Monocyte-conditioned medium contained heat-labile product(s) which also, although to a much lesser extent than intact monocytes, enhanced endothelial PAI-1 release. Similarly, monocytes cultured on top endothelial cell separated by a microporous filter enhanced the release of PAI-1 to a lesser extent. Thus, these findings indicate that monocytes enhance endothelial PAI-1 release by mechanisms that are, at least in part, dependent on cell-cell contact.

Pretreatment of endothelial cells with cytokines enhances the adherence of leukocytes, a process that is mediated by surface proteins expressed on both cell types. A three-dimensional model system for the simultaneous determination of leukocyte adherence and migration was used to study the contribution of CD11/CD18, endothelial leukocyte- adhesion molecule-1 (ELAM-1) and VLA-4 in neutrophil and monocyte adherence to and migration through cytokine-activated endothelial cells. Pretreatment of endothelial cells for 4 hours with recombinant interleukin-1 beta (rIL-1 beta) was found to enhance neutrophil adherence and migration to a much greater extent than monocyte adherence and migration. Neutrophil adherence was almost completely prevented by the combined use of monoclonal antibodies (MoAbs) against ELAM-1 and CD18. Although ELAM-1 has been designated an endothelial cell-specific cytokine-inducible receptor for neutrophils, we observed that ENA2, an anti-ELAM-1 MoAb, significantly reduced monocyte adherence about 30%. MoAbs against VLA-4, the ligand of the cytokine- inducible receptor VCAM-1, did not affect monocyte adherence. However, the combined use of the MoAbs against CD18, ELAM-1, and VLA-4 had a very strong and additive inhibitory effect on rIL-1 beta-induced monocyte adherence. The anti-CD18 MoAb reduced both rIL-1 beta-induced neutrophil and monocyte migration far below the level of the unstimulated controls, whereas neither the anti-ELAM-1 nor the anti-VLA- 4 MoAb significantly affected the process of migration. Our results indicate that neutrophils and monocytes initially adhere to cytokine- activated endothelial cells by CD18-independent and (to a lesser extent) by CD18-dependent mechanisms and subsequently change gears to a completely CD18-dependent migratory mechanism.

In this report we show that human endothelial cells (EC) can be detected in circulating blood by means of the EC-specific monoclonal antibody (MoAb) designated as CLB-HEC 19 and expressed quantitatively as number of cells per milliliter of whole blood. We first developed a method that was able to recover cultured human EC added to whole blood by Percoll density gradient centrifugation. The final recovery of the EC was 91.6% (SE = 0.65%). The EC were identified in the gradient subfractions by indirect immunofluorescence with the MoAb CLB-HEC 19. This method was then applied to the separation and characterization of EC or EC remnants from the whole arterial and venous blood taken from two groups of patients subjected to heart catheterization. Firstly, a preliminary blood screening of random samples was performed in a group of eight patients (group I) using a scoring evaluation for the presence of EC and the results were expressed as positivity index. Secondly, the complete blood screening of a group of ten patients (group II) was performed for the detection of immunofluorescent cells and the results were expressed as number of EC per milliliter of whole blood. Our results show in both group I and II a significant presence of EC in the blood after catheterization compared with their basal values. The minimal detectable concentration of EC was 0.06 cells/mL (SE = 0.057) of whole blood. We consider this technique as a suitable clinical test for the detection of EC injury in cardiovascular pathology.

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the effects of the interaction between human monocytes and endothelial cells on the production of type 1 plasminogen activator inhibitor (PAI-1) by endothelial cells. The effects of adherence and transendothelial migration of monocytes on endothelial PAI-1 release were compared with those of other leukocytes, conditioned media from monocytes, and interleukin-1 beta (IL-1 beta). Because the cell culture system used allows simultaneous analysis of the lumenal and the subendothelial compartment of endothelial cell monolayers, we also studied into which direction PAI-1 is released by endothelial cells. Under quiescent conditions, the net amount of PAI-1 accumulated at the lumenal side was twofold higher than that accumulated at the subendothelial side (about 2.0 micrograms PAI-1/10(6) cells and 1.1 microgram PAI-1/10(6) cells, respectively, in 24 hours), as analyzed by a quantitative immunoradiometric assay (IRMA). Direct cell-cell contact between highly purified monocytes and endothelial cells strongly enhanced the PAI-1 release by endothelial cells in a dose-dependent way, whereas lymphocytes and neutrophils did not affect endothelial PAI- 1 production. The monocyte-mediated increase was first detected after 12 hours of incubation and lasted for at least 48 hours. In the presence of two monocytes per endothelial cell, the increases of PAI-1 at the lumenal side and at the subendothelial side were 87% and 32% in 24 hours, respectively. The effect of IL-1 beta on PAI-1 release by endothelial cells closely resembled that observed for monocytes. Monocyte-conditioned medium contained heat-labile product(s) which also, although to a much lesser extent than intact monocytes, enhanced endothelial PAI-1 release. Similarly, monocytes cultured on top endothelial cell separated by a microporous filter enhanced the release of PAI-1 to a lesser extent. Thus, these findings indicate that monocytes enhance endothelial PAI-1 release by mechanisms that are, at least in part, dependent on cell-cell contact.

The cytoadhesin family consists of platelet glycoprotein (GP) IIb-IIIa and the endothelial vitronectin receptor. The beta subunit (GP IIIa) of these complexes expresses the alloantigen Zwa (or PIA1). This alloantigen is not expressed by members of other integrin subfamilies. By using immunoprecipitation and immunoblot techniques, we found that the beta subunit of a heterodimer, expressed by cultured human arterial smooth muscle cells and cultured foreskin fibroblasts, carries the Zwa antigenic determinant. Furthermore, the mobilities of the alpha and beta subunits of these two heterodimers are indistinguishable from those of the alpha and beta subunits of the endothelial vitronectin receptor. Therefore, we propose that the smooth muscle cell and fibroblast heterodimer are members of the cytoadhesin family. In Glanzmann's thrombasthenia, platelet GP IIb-IIIa is absent or severely reduced. Previously, we showed that endothelial cells from a thrombasthenic patient normally synthesize and express a GP IIb-IIIa- related molecule (the vitronectin receptor). Here we show that arterial smooth muscle cells, obtained from the same patient, express a surface molecule indistinguishable from the endothelial vitronectin receptor. We also demonstrate that both the endothelial and the smooth muscle cell GP IIIa-related molecule in this Glanzmann patient express Zwa. Our data indicate that (a) GP IIb-IIIa-related molecules on cell types other than platelets and endothelial cells can express Zwa in vitro, and (b) patients with Glanzmann's disease can express the Zwa antigen. This study substantiates our view that the defect in Glanzmann's disease is restricted to the megakaryocytes/platelets.

Endothelial cells express surface molecules that are involved in cell- matrix interaction, including the vitronectin receptor and the fibronectin receptor, both members of a family of cell adhesion receptors (integrins). Here we provide evidence that endothelial cells express a membrane molecule, indistinguishable from the platelet VLA-2 complex, which is a collagen receptor and a member of the integrin family. To identify this endothelial molecule, we have used a monoclonal antibody, CLB-10G11, which recognizes the VLA-2 complex from platelets. The molecule recognized by CLB-10G11 from endothelial cells was characterized as follows. (1) The monoclonal antibody precipitated two proteins from surface-labeled endothelial cells that corresponded to the platelet VLA-2 subunits (glycoprotein Ia and IIa) as judged by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional nonreduced/reduced SDS- PAGE. (2) Preclearing of endothelial cells with monoclonal antibody A- 1A5, an antibody that is directed against the common VLA beta subunit, removed all the CLB-10G11-binding material. (3) Crossed immunoelectrophoresis revealed that CLB-10G11 recognizes a single precipitation arc from either platelets or endothelial cells. Analysis of these two cell types in one gel again revealed one precipitation arc. The antigen of either cell type, recognized by CLB-10G11 could be precipitated by either polyclonal antiplatelet or polyclonal antiendothelial cell antiserum. Hence, it appears that endothelial cells express at least three different surface molecules (the vitronectin receptor, the fibronectin receptor and a collagen receptor), which may play an important role in controlling the anchorage of endothelial cells to the extracellular matrix.

Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) IIb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesized a membrane protein complex similar to the platelet GP IIb/IIIa complex. In this article, we demonstrate that endothelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP IIb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an “endotheliopathy.”

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A- peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.

A monoclonal antibody directed against the von Willebrand factor moiety (vWF) of factor VIII-von Willebrand factor (FVIII-vWF), which blocks ristocetin-induced platelet aggregation as well as the binding of FVIII- vWF to platelets in the presence of ristocetin, inhibited platelet adherence to human artery subendothelium when present in normal flowing blood. This monoclonal antibody, CLB-RAg 35, inhibited platelet adherence as a function of the shear rate. At wall shear rates below 500 s-1, platelet adherence was not affected, but at higher shear rates platelet adherence was gradually inhibited, reaching an average of 11% of the normal value at 2,500 s-1. Indirect immunofluorescence established the reactivity of CLB-RAg 35 with vWF present in artery subendothelium. Pretreatment of normal vessel walls with this antibody inhibited adherence of platelets in blood from a patient with severe homozygous von Willebrand's disease and in blood from normal individuals. The inhibition was shear-rate dependent and significant at high shear rates (2,500 s-1). By adding increasing amounts of purified FVIII-vWF to normal blood, the inhibition was gradually overcome. These data indicate that vWF present in the vessel wall contributes appreciably to platelet adherence. At high wall shear rates, platelet adherence is mediated virtually completely by both plasma FVIII-vWF and vWF in the vessel wall. At low wall shear rates (below 500 s-1), platelet adherence occurs independent of FVIII-vWF in plasma and vWF in the vessel wall.

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) lla. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP llb/GP IIIa complex. This evidence is based on the following observations: (1) Cl7, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (36S-methionine) as well as surface 125l-labeled cultured human endothelial cells; (2) upon crossed Immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125l-labeled C17 IgG, a single precipitate of the protein(s) recognized by Cl7 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-perfor-mance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A-peptide material and of fibrinogen in plasma as determined by HPLC (r= .88, P< .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P< .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.

Glanzmann's thrombasthenia is a bleeding disorder, inherited in an autosomal recessive way and characterized by an absence or deficiency of the platelet glycoprotein (GP) Ilb/IIIa complex. Recently, we and others demonstrated that cultured human umbilical vein endothelial cells synthesize a membrane protein complex similar to the platelet GP Ilb/IIIa complex. In this article, we demonstrate that endo- thelial cells isolated from the umbilical vein of a newborn with Glanzmann's thrombasthenia, as compared with normal endothelial cells, show no difference in their ability to synthesize and express this GP Ilb/IIIa complex. Our results indicate that Glanzmann's thrombasthenia is not accompanied by an “endotheliopathy.” © 1987 by Grune & Stratton, Inc.

Endothelial cells express surface molecules that are involved in cell-matrix interaction, including the vitronectin receptor and the fibronectin receptor, both members of a family of cell adhesion receptors (integrins). Here we provide evidence that endothelial cells express a membrane molecule, indistinguishable from the platelet VLA-2 complex, which is a collagen receptor and a member of the integrin family. To identify this endothelial molecule, we have used a monoclonal antibody, CLB-10G11, which recognizes the VLA-2 complex from platelets. The molecule recognized by CLB-10G11 from endothelial cells was characterized as follows. (1) The monoclonal antibody precipitated two proteins from surface-labeled endothelial cells that corresponded to the platelet VLA-2 subunits (glyco-protein la and lla) as judged by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional nonreduced/reduced SDS- PAGE. (2) Preclearing of endothelial cells with monoclonal antibody A-1A5, an antibody that is directed against the common VLA β subunit, removed all the CLB-10G11-binding material. (3) Crossed immunoelectrophoresis revealed that CLB-10G11 recognizes a single precipitation arc from either platelets or endothelial cells. Analysis of these two cell types in one gel again revealed one precipitation arc. The antigen of either cell type, recognized by CLB-10G11 could be precipitated by either polyclonal antiplatelet or polyclonal antiendothelial cell antiserum. Hence, it appears that endothelial cells express at least three different surface molecules (the vitronectin receptor, the fibronectin receptor and a collagen receptor), which may play an important role in controlling the anchorage of endothelial cells to the extracellular matrix.

The results of studies with cultured endothelial cells have shown that most von Willebrand factor (vWF) synthesized is directly secreted (constitutive pathway) and consists of both mature vWF, its precursor molecule pro-vWF, and the cleaved vWF prosequence. Only fully processed, functionally mature vWF is stored within the cell, together with the propeptide, and leaves the cell only on stimulation (regulated secretion). Both in resting and stimulated cultured endothelial cells, the stoichiometry of the released propeptide to the released mature vWF is essentially equimolar. In the present study, we have measured the molar ratio of propeptide to mature vWF in vivo, both under resting conditions and conditions that reflect activation of the endothelium. To this end, we devised a method that allows the measurement of the propeptide (vW antigen II) on a quantitative, ie, molar basis, using purified recombinant propeptide as a standard. Our results show that the molar concentration of the propeptide in normal plasma is about one tenth of the concentration of mature vWF (expressed as half-dimer concentration). This ratio is approximately 1:1 in the medium of cultured endothelial cells. On administration in healthy subjects of either 1-deamino-8-D-arginine vasopressin or endotoxin, both agents being known to elicit an intravascular increase of vWF, the molar ratio of propeptide to mature vWF increased fourfold to fivefold. The propeptide concentration returned to baseline values after about 6 to 7 hours of injection of each stimulus, whereas the increase of mature vWF was much more sustained. Because the respective half-lives of mature vWF and its propeptide clearly differ, measurement of the concentration of these proteins could provide a means to assess the extent of activation of the endothelium under physiological and pathophysiological conditions.

Since dialysis of human factor VIII against buffers of low ionic strength yielded two distinct components, and since the factor VIII fraction isolated from normal plasma showed von Willebrand factor activity as defined by the corrective effect on abnormal platelet retention and ristocetin aggregation in von Willebrand's disease, the present studies were performed to determine if the correcting activities could be attributed to one or both of the components. Dialysis of factor VIII against buffers of low ionic strength led, however, to a decrease in factor VIII procoagulant activity and the reduction of the correcting activities, which suggested that the intact aggregate was required for procoagulant activity and for von Willebrand factor activity. In this respect dialysis of factor VIII at low ionic strength differed from dissociation at high salt concentrations. The two low ionic strength components were identified by the use of a rabbit antiserum against factor VIII, and could be distinguished on the basis of specific antigenic structures. Dialysis of factor VIII at low ionic strength led to a decrease in antigenic determinants closely related to factor VIII function. Specific antibodies to the low ionic strength components inhibited factor VIII activity in normal plasma, but the residual factor VIII was higher than that after inhibition with antibodies against intact factor VIII. Both antibodies interfered with von Willebrand factor activity.

Hereditary von Willebrand factor (vWF ) deficiency in Dutch Kooiker dogs, which have undetectable levels of vWF, causes spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. Therefore, we used this canine model to study the in vivo effects of a new recombinant von Willebrand factor (rvWF ) preparation containing all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF ) and with a plasma-derived factor VIII/vWF concentrate (pdvWF ). In the vWF-deficient dogs, the half-life of vWF:Ag was 21.6 and 22.1 hours for rvWF, 7.7 hours for pdvWF, and 9 hours for LMW-rvWF; in vivo recovery of vWF:Ag was 59%, 64%, and 70% for rvWF, 33% for pdvWF and 92% for LMW-rvWF; in vivo recovery of RCoF was 78%, 110%, and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII, which were sustained even when vWF:Ag had decreased to nearly undetectable levels and only monomeric or dimeric species were detectable on agarose gels. At the dosages used, no effect was seen on bleeding time, but the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

The development of inhibitory antibodies to factor VIII in patients affected by a mild form of hemophilia A (factor VIII < 0.05 IU/mL) is considered a rare event. In this study, we evaluated the relationship between genotype and anti-factor VIII antibody formation in a patient with mild hemophilia A. Mutation analysis showed that a missense mutation in the factor VIII gene leading to replacement of Arg593 by Cys in the A2 domain of factor VIII was associated with hemophilia A in this patient. The anti-factor VIII antibodies present in the patient's plasma were characterized using metabolically labeled factor VIII fragments expressed in insect cells. The anti-factor VIII antibodies, composed of subclasses IgG2 and IgG4, reacted with both the fragment corresponding to the factor VIII heavy chain and the A2 domain. The Arg593 → Cys substitution was introduced into the cDNA encoding the A2 domain of factor VIII and the resulting construct was expressed in insect cells. Strikingly, the metabolically labeled A2 domain carrying the Arg593 → Cys mutation was not recognized by the anti-factor VIII antibodies present in the plasma of the patient. These data indicate that the anti-factor VIII antibodies are exclusively directed against exogenous factor VIII. This strongly suggests that the Arg593 → Cys substitution results in recognition of wild-type factor VIII as nonself and is thereby related to the formation of anti-factor VIII antibodies after factor VIII replacement therapy in this particular patient.

Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa–factor VIIIa complex assembly.

Hereditary von Willebrand factor (vWF ) deficiency in Dutch Kooiker dogs, which have undetectable levels of vWF, causes spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. Therefore, we used this canine model to study the in vivo effects of a new recombinant von Willebrand factor (rvWF ) preparation containing all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF ) and with a plasma-derived factor VIII/vWF concentrate (pdvWF ). In the vWF-deficient dogs, the half-life of vWF:Ag was 21.6 and 22.1 hours for rvWF, 7.7 hours for pdvWF, and 9 hours for LMW-rvWF; in vivo recovery of vWF:Ag was 59%, 64%, and 70% for rvWF, 33% for pdvWF and 92% for LMW-rvWF; in vivo recovery of RCoF was 78%, 110%, and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII, which were sustained even when vWF:Ag had decreased to nearly undetectable levels and only monomeric or dimeric species were detectable on agarose gels. At the dosages used, no effect was seen on bleeding time, but the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

The cytoadhesin family consists of platelet glycoprotein (GP) IIb-IIIa and the endothelial vitronectin receptor. The β subunit (GP IIIa) of these complexes expresses the alloan-tigen Zwa(or PIA1). This alloantigen is not expressed by members of other integrin subfamilies. By using immuno-precipitation and immunoblot techniques, we found that the β subunit of a heterodimer, expressed by cultured human arterial smooth muscle cells and cultured foreskin fibroblasts, carries the Zwaantigenic determinant. Furthermore, the mobilities of the aand βsubunits of these two heterodimers are indistinguishable from those of the aand βsubunits of the endothelial vitronectin receptor. Therefore, we propose that the smooth muscle cell and fibroblast heterodimer are members of the cytoadhesin family. In Glanzmann's thrombasthenia, platelet GP IIb-IIIa is absent or severely reduced. Previously, we showed that endothe- lial cells from a thrombasthenic patient normally synthesize and express a GP IIb-IIIa-related molecule (the vitronectin receptor). Here we show that arterial smooth muscle cells, obtained from the same patient, express a surface molecule indistinguishable from the endothelial vitronectin receptor. We also demonstrate that both the endothelial and the smooth muscle cell GP IIIa-related molecule in this Glanzmann patient express Zwa. Our data indicate that (a) GP IIb-IIIa-related molecules on cell types other than platelets and endothelial cells can express Zwa in vitro, and (b) patients with Glanzmann's disease can express the Zwa. antigen. This study substantiates our view that the defect in Glanzmann's disease is restricted to the megakaryo-cytes/platelets.

The development of inhibitory antibodies to factor VIII in patients affected by a mild form of hemophilia A (factor VIII < 0.05 IU/mL) is considered a rare event. In this study, we evaluated the relationship between genotype and anti-factor VIII antibody formation in a patient with mild hemophilia A. Mutation analysis showed that a missense mutation in the factor VIII gene leading to replacement of Arg593by Cys in the A2 domain of factor VIII was associated with hemophilia A in this patient. The anti-factor VIII antibodies present in the patient's plasma were characterized using metabolically labeled factor VIII fragments expressed in insect cells. The anti-factor VIII antibodies, composed of subclasses IgG2 and IgG4, reacted with both the fragment corresponding to the factor VIII heavy chain and the A2 domain. The Arg593→ Cys substitution was introduced into the cDNA encoding the A2 domain of factor VIII and the resulting construct was expressed in insect cells. Strikingly, the metabolically labeled A2 domain carrying the Arg593→ Cys mutation was not recognized by the anti-factor VIII antibodies present in the plasma of the patient. These data indicate that the anti-factor VIII antibodies are exclusively directed against exogenous factor VIII. This strongly suggests that the Arg593→ Cys substitution results in recognition of wild-type factor VIII as nonself and is thereby related to the formation of anti-factor VIII antibodies after factor VIII replacement therapy in this particular patient.

During Plasmodium falciparum malaria infections, von Willebrand factor (VWF) levels are elevated, postmortem studies show platelets colocalized with sequestered infected erythrocytes (IEs) at brain microvascular sites, whereas in vitro studies have demonstrated platelet-mediated IE adhesion to tumor necrosis factor-activated brain endothelium via a bridging mechanism. This current study demonstrates how all these observations could be linked through a completely novel mechanism whereby IEs adhere via platelet decorated ultra-large VWF strings on activated endothelium. Using an in vitro laminar flow model, we have demonstrated tethering and firm adhesion of IEs to the endothelium specifically at sites of platelet accumulation. We also show that an IE pro-adhesive state, capable of supporting high levels of binding within minutes of induction, can be removed through the action of the VWF protease ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). We propose that this new mechanism contributes to sequestration both independently of and in concert with current adhesion mechanisms.

During Plasmodium falciparummalaria infections, von Willebrand factor (VWF) levels are elevated, postmortem studies show platelets colocalized with sequestered infected erythrocytes (IEs) at brain microvascular sites, whereas in vitro studies have demonstrated platelet-mediated IE adhesion to tumor necrosis factor-activated brain endothelium via a bridging mechanism. This current study demonstrates how all these observations could be linked through a completely novel mechanism whereby IEs adhere via platelet decorated ultra-large VWF strings on activated endothelium. Using an in vitro laminar flow model, we have demonstrated tethering and firm adhesion of IEs to the endothelium specifically at sites of platelet accumulation. We also show that an IE pro-adhesive state, capable of supporting high levels of binding within minutes of induction, can be removed through the action of the VWF protease ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). We propose that this new mechanism contributes to sequestration both independently of and in concert with current adhesion mechanisms.

No relevant conflicts of interest to declare.

Toxoplasma gondiiis a ubiquitous protozoan parasite which infects approximately one third to one half of the world's population. In the immunocompetent host it typically causes a self limiting and asymptomatic infection before entering a lifelong latent phase. Reactivation and disseminated toxoplasmosis occur in the setting of impaired cellular immunity as described in patients with acquired immunodeficiency syndrome (AIDS) secondary to human immunodeficiency virus (HIV) infection and those receiving prolonged immunosuppression post solid organ or allogeneic haematopoietic stem cell transplantation. Toxoplasmosis post autograft has been rarely described. We present a case of toxoplasma encephalitis in a patient with persistent CD4 lymphopenia post autograft for peripheral T-cell lymphoma (PTCL).

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IE) to activated endothelial cell (EC) surfaces plays a key role in the pathophysiology of cerebral malaria. Moreover, recent evidence suggests that platelet adhesion and aggregation are critical in facilitating this cytoadhesion process. We have recently reported marked increased plasma VWF and VWF propeptide levels in severe P. falciparum infection, indicative of acute EC perturbation. Furthermore, plasma VWF:Ag levels in patients with malaria inversely correlated with platelet count, and plasma VWF propeptide levels correlated with other established biochemical markers of malaria severity, including plasma lactate. Nevertheless, it remains unclear whether VWF plays any direct role in mediating IE cytoadhesion, or whether the increased plasma levels of both VWF and propeptide merely serve as a marker of acute EC activation. To address this question, we collected plasma samples from a cohort of African children presenting with cerebral malaria (CM), or non-cerebral severe malaria (SM). In children with CM and SM, plasma VWF:Ag levels were significantly elevated (medians 3.1 and 3.4 IU/ml) as before. VWF collagen binding (VWF:CB) levels were also markedly increased (medians 7.6 and 7.0 IU/ml). Furthermore, the relative rise in VWF:CB was much greater, so that the ratio of CB:Ag was consistently increased. VWF multimer analysis demonstrated abnormal circulating ULVWF multimers in children with CM and SM, consistent with acute, regulated VWF secretion. To characterize mechanisms responsible for the markedly increased VWF:CB and circulating ULVWF in malarial plasma, we investigated ADAMTS13 antigen and activity levels in CM and SM plasma. Plasma ADAMTS13 activity levels (FRETS-VWF73 assay) and antigen levels were both significantly reduced (medians = 0.63 U/ml and 0.56 U/ml; p< 0.001) in children with CM and SM compared to controls. Classical mixing studies of malaria and normal plasma demonstrated no evidence of immediate ADAMTS13 inhibition. However, ADAMTS13 activity in normal plasma was significantly reduced (~60%) following 30 min incubation with malarial plasma (75%:25% mix). This inhibitory ability of malaria plasma was further confirmed by spiking malarial plasmas with recombinant human ADAMTS13. Significant time-dependent inhibition of FRETS-VWF73 activity was again observed in the plasmas of children with severe P. falciparum, but not in normal control plasmas. Potential inhibitors of ADAMTS13 in vivo include interleukin-6 (IL-6), thrombospondin-1 (TSP-1), thrombin and plasmin, free plasma haemoglobin, and reduced FVIII levels. Although plasma IL-6 levels were significantly elevated in children with either CM (mean 240 pg/ml; p<0.001) or SM (mean 217 pg/ml; p=0.01) compared to normal controls, levels did not approach those previously reported necessary to inhibit ADAMTS13 activity. Disseminated intravascular coagulation (DIC) is associated with enhanced thrombin generation, and consumption of FVIII, both of which can inhibit VWF proteolysis by ADAMTS13. However, in children with CM or SM respectively, we observed significantly increased plasma FVIII:C levels. Although intravascular haemolysis is also a recognised complication of malarial infection, we observed only minor increased plasma haemoglobin concentrations, again well below that previously described to significantly inhibit ADAMTS13 activity. Finally, in contrast to the increased plasma levels of IL-6 and FVIII:C, we found that plasma TSP-1 levels were not significantly elevated in children with either CM or SM compared to pooled normal plasma. In conclusion, we demonstrate that the presence of ULVWF multimers in the plasma of children with severe P. falciparum malaria is the result of acute EC activation and release of ULVWF from WP bodies; significantly reduced plasma ADAMTS13 antigen levels a circulating but unidentified inhibitor of human ADAMTS13 activity. In view of the critical role played by VWF in mediating platelet adhesion/aggregation, and the accumulating evidence suggesting that platelet adhesion/aggregation also facilitate cytoadhesion of IE, we propose a novel role for ULVWF multimers in the pathophysiology of severe P. falciparum malaria.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IE) to activated endothelial cell (EC) surfaces plays a key role in the pathophysiology of cerebral malaria. Moreover, recent evidence suggests that platelet adhesion and aggregation are critical in facilitating this cytoadhesion process. We have recently reported marked increased plasma VWF and VWF propeptide levels in severe P. falciparuminfection, indicative of acute EC perturbation. Furthermore, plasma VWF:Ag levels in patients with malaria inversely correlated with platelet count, and plasma VWF propeptide levels correlated with other established biochemical markers of malaria severity, including plasma lactate. Nevertheless, it remains unclear whether VWF plays any direct role in mediating IE cytoadhesion, or whether the increased plasma levels of both VWF and propeptide merely serve as a marker of acute EC activation.

Background: The antiphospholipid syndrome is characterized by the occurrence of vascular thrombosis combined with the presence of antiphospholipid antibodies (aPL) in plasma of patients. Recently it was published that aPL with lupus anticoagulant activity (LAC), caused by anti-beta2-glycoprotein I (beta2GPI) antibodies, highly correlate with a history of thrombosis. aPL-related resistance against activated protein C (APC) is one of the proposed mechanism responsible for thrombosis. We investigated a possible correlation between a beta2GPI-dependent LAC and increased APC-resistance in a population of 22 plasma samples with LAC activity. Methods: Twenty-two LAC-positive plasma samples were tested for beta2GPI-dependence (titration of cardiolipin into an APTT-based assay), increased APC-resistance, anti-beta2GPI IgG/IgM antibodies, anti-prothrombin IgG/IgM antibodies and anti-protein C IgG/IgM antibodies. In addition, a monoclonal anti-beta2GPI antibody and patient-purified IgG (both with LAC activity) were diluted in plasma with/without protein C and tested for occurrence of a beta2GPI-dependent LAC (normalization of clotting time by the addition of cardiolipin). To study aPL-induced APC-resistance in more detail, surface plasmon resonance analysis was used to investigate binding between APC and beta2GPI in the presence/absence of a mouse-derived monoclonal anti-beta2GPI antibody. Results: Eleven plasma samples that displayed a beta2GPI-dependent LAC also showed increased APC resistance. In contrast, only 1 of the 11 plasma samples with a beta2GPI-independent LAC displayed increased APC-resistance. None of the other serological parameters (antibodies against beta2-glycoprotein I, prothrombin or protein C) displayed the same association with increased APC resistance as a beta2-glycoprotein I dependent LAC. Furthermore, we found a linear correlation between the potency of a beta2GPI-dependent LAC and the level of APC-resistance. When a monoclonal anti-beta2GPI antibody and a patient-purified IgG were tested for a beta2GPI-dependent LAC, both antibodies did not display a beta2GPI-dependent LAC when diluted in protein C deficient plasma. In literature it has been proposed that direct binding of beta2GPI to APC results in a decreased activity of APC. By using surface plasmon resonance analysis, we found that beta2GPI displayed a higher affinity for coated APC in the presence of the monoclonal anti-beta2GPI antibody (4 nM) compared to beta2GPI alone (400 nM). Conclusion: The results of this study indicate that by adding cardiolipin into an APTT-based clotting assay, one can detect beta2GPI-dependent LAC based on increased resistance against APC. Increased resistance against activated protein C might result from direct binding of beta2GPI to activated protein C. In conclusion, our observations indicate a direct correlation between a major clinical symptom of APS (thrombosis), a diagnostic assay (beta2GPI-dependent LAC) and a potential mechanism responsible for thrombosis in the antiphospholipid syndrome (increased APC-resistance).

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.

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.

Background: The antiphospholipid syndrome is characterized by the occurrence of vascular thrombosis combined with the presence of antiphospholipid antibodies (aPL) in plasma of patients. Recently it was published that aPL with lupus anticoagulant activity (LAC), caused by anti-beta2-glycoprotein I (beta2GPI) antibodies, highly correlate with a history of thrombosis. aPL-related resistance against activated protein C (APC) is one of the proposed mechanism responsible for thrombosis. We investigated a possible correlation between a beta2GPI-dependent LAC and increased APC-resistance in a population of 22 plasma samples with LAC activity.

Acquired thrombotic thrombocytopenic purpura (TTP) is often accompanied by the presence of (auto-) antibodies directed against the plasma metalloprotease ADAMTS13. The antibodies prevent efficient proteolytic cleavage of unusually large von Willebrand factor multimers by ADAMTS13, which is highly reactive with platelets. This may result in the formation of platelet aggregates that occlude the microvasculature of patients with TTP. In all presently characterized patients with acquired TTP and detectable anti-ADAMTS13 antibodies, antibodies directed to the (cysteine-rich) spacer domain were present. In order to study anti-ADAMTS13 antibodies at the molecular level, we have isolated single-chain Fv antibody fragments (scFv) from an immunoglobulin V-gene phage-display library derived from CD19+ B-cells of a patient with acquired TTP. From this library scFv I-9 was isolated, of which the variable heavy chain segment is homologous to members of the VH1-69 family. Using ELISA and immunoprecipitation analysis we confirmed that antibody I-9 also binds to the spacer domain. We converted the scFv antibody fragment into a full-length human antibody to better study its effect on proteolytic cleavage of VWF and substrate binding. Antibody I-9 was included into a modified FRETS-VWF73 assay and a dose-dependent inhibition of VWF73 proteolysis by ADAMTS13 was observed. In addition, antibody I-9 was able to inhibit the proteolysis of plasma-derived VWF multimers by ADAMTS13. In both assays inhibition was not very efficient; which is most likely due to the low affinity of antibody I-9 for ADAMTS13. Chimeric ADAMTS13 variants in which 5–10 amino acid residues of the ADAMTS13 spacer domain have been exchanged for the corresponding sequence of ADAMTS1 were used to determine the binding site for antibody I-9 in the spacer domain. Our findings show that amino acid regions 572–579 and 657–666 of the spacer domain are required for binding of antibody I-9, although several other regions are also involved in the interaction. We have previously shown that these amino acid regions are also crucial for binding of (polyclonal) antibodies present in plasma of patients with acquired TTP. Additionally, competition experiments were performed to address whether antibodies with similar properties as antibody I-9 are indeed present in plasma from patients with acquired TTP during acute disease. Plasma from 6 patients with acquired TTP was able to compete in a dose-dependent manner with antibody I-9 for binding to immobilized ADAMTS13, including plasma from the patient whose lymphocytes were used for construction of the phage-display library. These results show that antibodies with similar epitope-specificity as antibody I-9 are present in plasma of patients with acquired TTP. Since anti-spacer domain antibodies have been found in all presently characterized patients with anti-ADAMTS13 antibodies, the current data suggest that antibody I-9 is representative of the pathogenic antibodies present in plasma of the majority of patients with acquired TTP.

Inherited thrombophilia is known to increase the risk for venous thromboembolism (VTE). Furthermore, an association between recurrent pregnancy loss and hypertensive pregnancy complications (including preeclampsia and HELLP syndrome) appears present. A relationship between thrombophilia and arterial cardiovascular disease could not be demonstrated in most studies. Since thrombophilia is prevalent in patients with VTE, testing for these abnormalities often reveals positive test results which may lead to widespread testing. This is also the case in patients with various other conditions. However, there is little convincing evidence about effectiveness of testing due to the uncertainty with respect to therapeutic consequences of a positive thrombophilia test results. To assess the current practice of thrombophilia testing in The Netherlands and to get an impression of the therapeutic implications of testing for patients, simple questionnaires were sent out to physicians that ordered tests for protein S, C, or antithrombin activity, factor V Leiden (or APC resistance) or the prothrombin 20210A mutation in 1998 consecutive individuals who were investigated between November 2003 and March 2004. The tests were performed in a laboratory that serves as a diagnostic facility for mainly non-academic hospitals and general practitioners throughout the Netherlands. Of a total of 1265 returned questionnaires, 1130 were suitable for analysis (response rate 63%). Of the tested individuals, 64% were female (median age 38 years, inter-quartile-range [IQR] 30–50) and 35% were male (median age 51, IQR 41–59). Tests were ordered by internists (37%), gynaecologists (20%), neurologists (15%) and general practitioners (13%). Only 42% of the tested individuals had experienced VTE, 23% arterial cardiovascular disease, and 17% had had pregnancy complications. A known carrier or VTE in the family was the reason for testing in 180 asymptomatic individuals (16%). Testing had had no therapeutic consequences in 869 tests (77%). In 32% of these patients, physicians stated that had the test revealed a thrombophilic defect, this would not have altered management of the patient. This study shows that testing for thrombophilia occurs very often in patients with various conditions. In the majority of cases, the results do not change therapeutic management of tested patients. Widespread thrombophilia testing is costly and may cause unnecessary concern in carriers. This study underscores the need to acquire high level evidence about its effectiveness that should include clinical outcomes as well as quality of life and costs.