The spider hemolymph clot proteome reveals high concentrations of hemocyanin and von Willebrand factor-like proteins.

Arthropods include chelicerates, crustaceans, and insects that all have open circulation systems and thus require different properties of their coagulation system than vertebrates. Although the clotting reaction in the chelicerate horseshoe crab (Family: Limulidae) has been described in details, the overall protein composition of the resulting clot has not been analyzed for any of the chelicerates. The largest class among the chelicerates is the arachnids, which includes spiders, ticks, mites, and scorpions. Here, we use a mass spectrometry-based approach to characterize the spider hemolymph clot proteome from the Brazilian whiteknee tarantula, Acanthoscurria geniculata. We focused on the insoluble part of the clot and demonstrated high concentrations of proteins homologous to the hemostasis-related and multimerization-prone von Willebrand factor. These proteins, which include hemolectins and vitellogenin homologous, were previously identified as essential components of the hemolymph clot in crustaceans and insects. Their presence in the spider hemolymph clot suggests that the origin of these proteins' function in coagulation predates the split between chelicerates and mandibulata. The clot proteome reveals that the major proteinaceous component is the oxygen-transporting and phenoloxidase-displaying abundant hemolymph protein hemocyanin, suggesting that this protein also plays a role in clot biology. Furthermore, quantification of the peptidome after coagulation revealed the simultaneous activation of both the innate immune system and the coagulation system. In general, many of the identified clot-proteins are related to the innate immune system, and our results support the previously suggested crosstalk between immunity and coagulation in arthropods.

Immunoexpression of MMP-9, VEGF, and vWF in central and peripheral giant cell lesions of the jaws.

BACKGROUND: Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) are pathological conditions of the jaws that share the same microscopic features, but differ clinically in terms of their behavior. Our aim was to compare the immunoexpression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in CGCL and PGCL, relating them to the angiogenic index. METHODS: Twenty CGCL and 20 PGCL were selected for analysis of the immunoexpression of MMP-9 and VEGF in multinucleated giant cells (MGC) and mononucleated cells (MC). Angiogenic index was determined by microvessel count (MVC) using anti-von Willebrand factor antibody. RESULTS: The CGCL showed slightly higher expression of MMP-9 than PGCL. In comparison with PGCL, the CGCL showed higher expression of VEGF both in MC (P < 0.05) and in total cells (P < 0.05). PGCL exhibited higher MVC than CGCL (P < 0.05). CONCLUSIONS: MMP-9 and VEGF might play an important role in the osteoclastogenesis process in CGCL. The higher MVC in PGCL might be related to the reactive nature of these lesions.

Clearance of von Willebrand factor.

The life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of the VWF gene in endothelial cells and megakaryocytes to the removal of VWF from the circulation. The various aspects of VWF clearance have been the objects of intense research in the last few years, stimulated by observations that VWF clearance is a relatively common component of the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, improving the survival of VWF is now considered as a viable therapeutic strategy to prolong the half-life of factor VIII in order to optimise treatment of haemophilia A. The present review aims to provide an overview of recent findings with regard to the molecular basis of VWF clearance. A number of parameters have been identified that influence VWF clearance, including its glycosylation profile and a number of VWF missense mutations. In addition, in-vivo studies have been used to identify cells that contribute to the catabolism of VWF, providing a starting point for the identification of receptors that mediate the cellular uptake of VWF. Finally, we discuss recent data describing chemically modification of VWF as an approach to prolong the half-life of the VWF/FVIII complex.

Thrombin-mediated in vitro processing of pro-von Willebrand factor.

Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-provWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Building on previous studies indicating that recombinant provWF when infused into animals can also be processed extracellularly in vivo, we investigated the processing of provWF in vitro. Incubation of a recombinant provWF (rpvWF) preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in provWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the provWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using 125-labeled provWF. Processing was facilitated by CaCl2 but prevented by a thrombin inhibitor and did not occur in prothrombin-depleted plasma. When recombinant provWF was incubated with increasing amounts of purified thrombin, the extent of provWF processing was dose-dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino terminal amino-acid analysis. Binding of provWF to collagen decreased the thrombin concentration necessary for propeptide removal to a concentration in the range of that found during blood clotting. Meizothrombin, an intermediate of prothrombin activation, was also able to induce dose-dependent removal of the propeptide from rpvWF. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused rpvWF suggesting that thrombin plays a part in the processing events in vivo.

Effect of polyvinyl chloride plastic on the growth and physiology of human umbilical vein endothelial cells.

In this work, human umbilical vein endothelial cells were cultured on common polystyrene cell culture plates, referred to as control plates, as well as on soft polyvinyl chloride plastics (PVC). Growth of human umbilical endothelial cells (HUVEC) on PVC coated with gelatin, collagen A and heparin plasma was significantly less than that on the control plates coated with the same substance or fibronectin. Cells cultured on PVC produced up to four times as much tissue plasminogen activator than control cells. With reference to plasminogen activator inhibitor 1 (PAI-1), more PAI-1 was released from cells grown on PVC than from those on the control plates coated with gelatin and collagen A. After endotoxin stimulation, the PAI-1 release of HUVEC cultured on PVC was significantly higher than that of control cells with the exception of cells grown on the fibronectin-coated PVC that showed no difference. It is concluded that the type of plastic and coat used to culture HUVEC play a definite role in their growth and function.

An ultrastructural and immunohistochemical study of human dental pulp: identification of Weibel-Palade bodies and von Willebrand factor in pulp endothelial cells.

Special and specific immunohistochemical techniques as well as routine transmission electron microscopy were used to identify the presence of von Willebrand factor (vWF), a blood clotting factor essential to normal hemostasis, and Weibel-Palade bodies (WPB's), respectively, in the endothelial cells lining the blood vessels from both normal and inflamed human pulpal tissues. In human endothelial cells, WPB's are peculiar and specialized organelles which store vWF. All classes of blood vessels (capillaries, arterioles, arteries, venules, and veins) were vWF positive. The fine structural studies showed similar results with regard to the presence of WPB's. Interestingly, morphometric analyses conducted on the same tissues using either light or transmission electron microscopy showed that significantly more vWF-positive blood vessels were seen in the inflamed tissues. In agreement with the latter observation, transmission electron microscopy showed that more vascular endothelial cells contained WPB's in the inflamed tissues when compared with the normal tissues. From this it appears that during pulpal inflammation, the cascade of events associated with hemostasis may be activated with the increased synthesis and release of vWF by endothelial cells.

Von Willebrand factor and autoantibodies against oxidized LDL in hemodialysis patients treated with vitamin E-modified dialyzers.

Oxidant stress is a well known cause of damage in the atherosclerotic process. Vitamin E is one of the most promising natural antioxidants. In this study we investigated if a vitamin E-coated dialyzer was able to reduce the plasma levels of auto-antibodies against oxidized-LDL, von Willebrand factor (vWf) and thrombomodulin (TM) as markers of endothelial damage. In this controlled 6-month prospective study, we investigated these markers in two matched groups (n=16 each) of patients on regular hemodialysis not yet diagnosed for atherosclerosis cardiovascular disease (ACVD) (mean age=58.3+/-7.0 yrs, mean dialysis age=30.1+/-10.0 months), in which cellulosic (CLS) and vitamin E-modified dialyzers (CLE) were compared. At inclusion all the patients were treated with CLS. Then, the study group was shifted to CLE for 6 months. At baseline the patients showed normal levels of vitamin E and high levels of oxLDL-Ab, vWf and TM compared to healthy subjects. In the CLE group oxLDL-Ab and vWf, but not TM levels, decreased progressively (from 472+/-287 to 264+/-199 mU/mL, p<0.0001 and from 101.1+/-7.5% to 76.7+/-18.5%; p<0.001, respectively), and vitamin E increased from 4.40+/-0.81 to 7.81+/-1.16 microg/mg of cholesterol. At the end of the study, 8 of the patients treated with CLE were randomly selected and went back to the membrane without Vitamin E for six months. They showed an significant increase in OxLDL-Ab and vWf levels and a significant reduction in tocoferol levels. In conclusion, CLE compared to cellulosic dialyzers can lower some indices of damage to LDL and endothelial cells.

Endothelial differentiation of human stem cells seeded onto electrospun polyhydroxybutyrate/polyhydroxybutyrate-co-hydroxyvalerate fiber mesh.

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues.

Retention test Homburg to monitor the adhesive properties of von Willebrand factor after substitution or desmopressin therapy.

To diagnose von Willebrand disease (vWD) and to monitor drug efficacy, several tests have been established that are not, however, focused on the platelet adhesion properties of von Willebrand factor (vWF). The new platelet retention test Homburg (RTH) is characterized by a nonthrombogenic filter that retains platelets from blood when it is pressed through this filter. It was the aim of this study to examine the capability of this test to monitor the adhesive properties of vWF after its substitution in vWD or its release by desmopressin infusion. The RTH demonstrated a striking sensitivity for vWF after its release from endogenous storage sites or its supplementation in vivo as well as in vitro, whereas it did not detect an inhibition of platelet aggregability due to aspirin. Desmopressin infusions led to an immediate and highly significant increase of RTH platelet retention, followed by a gradual decline to initial values during the next 4 hours. The transfusion of a vWF concentrate also resulted in RTH data that demonstrated different kinetics than established parameters such as vWF:antigen, vWF:RiCoF, or the PFA-100 in vitro bleeding analysis. Additional in vitro experiments confirmed the correlation of RTH values with vWF concentrations. In conclusion, the RTH may be efficient to complement presently used measures to monitor vWD therapy with desmopressin or vWF concentrates.

Molecular cloning, expression and assembly of multimeric von Willebrand factor.

Recently, substantial progress has been made in our knowledge of the domains involved in correlating structure and function of vWF, as well as in the biosynthesis and assembly of multimeric vWF. These studies were greatly supported by the development of three new techniques. (1) In vitro culturing of (human) vascular endothelial cells has allowed studies on the subcellular localization for dimerization of pro-vWF subunits, multimerization, carbohydrate and proteolytic processing. Moreover, this approach has provided insight into the complex intracellular routing of vWF that proceeds by either one of two pathways. During the constitutive pathway, vWF is packaged in secretory vesicles that are rapidly secreted both at the luminal and at the basolateral side. The regulated pathway includes storage of vWF molecules in specialized organelles, i.e. the Weibel-Palade bodies. (2) The application of proteases to dissect purified multimeric vWF and to assign function(s) to defined proteolytic fragments of vWF. Monoclonal antibodies, raised against native vWF, that block specific functions and bind to fragments are subsequently employed to correlate structure and function. More precise localizations of functions are now feasible, using overlapping synthetic peptides derived from the primary amino acid sequence of (pro)-vWF and antibodies raised against such peptides. This approach has permitted a fine mapping of the interaction site of vWF with the platelet receptor glycoprotein (GP) IIB/IIIA. (3) Molecular cloning of full-length vWF cDNA and the development of eukaryotic expression systems have substantially increased the possibilities to investigate structures on pro-vWF involved in the biosynthesis and the assembly of multimers. In particular, the construction of point and deletion mutants of vWF, employing vWF cDNA, and subsequent expression in heterologous cells have demonstrated that proteolytic processing of pro-vWF, between arginine (Arg-763) and serine (Ser-764), to generate free propolypeptide and mature vWF is not required for multimerization. Finally, the propolypeptide has an obligatory role for the formation of multimers, enabling interdimer disulphide bonding of free sulphhydryl groups located within the mature vWF part of pro-vWF.

Biosynthesis of human von Willebrand factor.

Endothelium forms the inner lining of all blood vessels and, as a consequence, is in direct contact with the blood. Because of this and the synthesis and secretion of hemostatic components, the endothelium is able to modulate coagulation and fibrinolysis. An important hemostatic factor synthesized by endothelial cells is the von Willebrand factor (vWF). vWF is a large plasma glycoprotein which promotes the adhesion of platelets to the vessel wall after a vascular injury. vWF is initially synthesized as a pre-pro-polypeptide. During its transport to the outside of the cell, the single-chain polypeptides are assembled into multimers. The pro-polypeptide can be cleaved and also be secreted. Free pro-polypeptide is identified as von Willebrand antigen II, a plasma glycoprotein of unknown function. Plasma vWF consists of a heterogenous series of multimers, composed of an apparently single-type glycoprotein subunit, linked together by disulfide bonds. The hemostatic potency of vWF was shown to increase with increasing multimer size. Therefore, the multimeric assembly of vWF is a crucial aspect in vWF biosynthesis. Furthermore, vWF synthesized by endothelial cells can either be secreted constitutively or stored and released upon stimulation of the endothelial cell. In this review, data are presented which contribute to the understanding of the biosynthetic pathway and complex processing which vWF has to undergo before it is secreted by the endothelial cell. These data have allowed a prediction of the sequential events underlying vWF biosynthesis, processing, multimer assembly, and secretion.

Proteolytic cleavage of the precursor of von Willebrand factor is not essential for multimer formation.

Monkey kidney cells (COS-1), transfected with full-length human von Willebrand factor (vWF) cDNA encoding the precursor of vWF (pro-vWF), mimic the characteristics of the biosynthesis and of the constitutive secretory pathway, displayed by cultured vascular endothelial cells. Such heterologous transfected cells are able to cleave pro-vWF, generating the propolypeptide and mature vWF, and to assemble pro-vWF dimers into a series of multimers, similarly to endothelial cells. Evidence is presented showing that proteolytic processing of pro-vWF by COS-1 cells occurs at the peptide bond between arginine and serine in the sequence Lys762-Arg763-Ser764, identical to endothelial cell-associated proteolysis. This conclusion stems from the observation that substitution of Arg763 by a glycine residue completely abolishes proteolytic processing. As a result, transfection of COS-1 with the mutant vWF-Gly763 cDNA does not significantly affect the multimeric organization of secreted vWF molecules. Consequently, we conclude that proteolytic processing of pro-vWF is not required for multimer formation. Pulse-chase labeling of COS-1 cells transfected with full-length vWF cDNA reveals pro-vWF exclusively in cell lysates, whereas both pro-vWF and mature vWF are encountered in the conditioned medium. These observations indicate that proteolytic processing of pro-vWF is a "late" event during intracellular routing of these molecules or may occur extracellularly.

Cytocompatibility study of NHLBI primary reference materials using human endothelial cells.

Four different materials, low density polyethylene (LDPE), polydimethylsiloxane (PDMS), polyvinylcholoride (PVC) and cellulose, were selected by the Devices and Technology Branch of the National Heart Lung and Blood Institute (NHLBI) as primary reference materials for blood contacting. Among the wide variety of tests proposed to assess hemocompatibility of short-term blood contacting catheters, it was desirable to rule out whether these materials could release toxics for vascular cells of the physiological environment. Thus, the cytocompatibility of these materials have been checked towards human umbilical vein endothelial cells: the method used avoids direct contact between cells and materials but evaluates the effect of possible toxic substances leached from materials. These substances were obtained under defined conditions according to a standard. The results show that the extracts of cellulose and LDPE provoke an important cytotoxic effect on the endothelial cell cultures, while the extracts of PDMS and PVC allow the obtention of endothelial cell lining of the reference surface, with a correct global metabolic activity and the intracellular presence of von Willebrand factor.

Human endothelial cell growth and coagulant function varies with respect to interfacial properties of polymeric substrates.

The in vitro coagulant function of human aortic endothelial cells (HAECs) was investigated when grown on a series of polymer surfaces that ranged from hydrophobic to hydrophilic. The polymer interface materials were prepared by radiofrequency plasma polymerization from hexamethyl-disilazane, gamma-butyrolactone, and N-vinyl-2-pyrrolidone and deposited onto tissue culture Permanox. The three plasma polymers were noncytotoxic. When precoated with fibronectin (FN), HAECs on all four polymer surfaces were similar with respect to cell proliferation and coagulant function. Without FN precoating, cell proliferation and spreading increased with increasing surface hydrophilicity. Normalized production of tissue-type plasminogen activator increased with increasing hydrophilicity of the polymers during early incubation times, as did tissue plasminogen activator/plasminogen activator inhibitor-1 ratios. In comparison, normalized von Willebrand factor release decreased on the more hydrophilic surfaces. Thus, both endothelial cell growth and some coagulant/fibrinolytic functions are improved with increasing substrate hydrophilicity.

Acute hyperglycemia and hyperinsulinemia enhance vasodilatation in Type 1 diabetes mellitus without increasing capillary permeability and inducing endothelial dysfunction.

Uncomplicated Type 1 (insulin-dependent) diabetes mellitus is characterized by generalized vasodilatation. Its possible correlates, increased microvascular permeability and endothelial dysfunction, have been associated with long-term complications. The objective was to study the effects of acute hyperglycemia and hyperinsulinemia, both separately and in combination, on skin microvascular flow, capillary permeability, capillary recruitment, and endothelial dysfunction in Type 1 diabetes mellitus. Sixteen Type 1 diabetic patients (all normoalbuminuric, no (pre-)proliferative retinopathy) underwent a euglycemic (glucose target 5.0 mmol/L, insulin infused at 30 mU x kg(-1) x h(-1)), a hyperglycemic (glucose target 12.0 mmol/L, insulin 30 mU x kg(-1) x h(-1)), a hyperinsulinemic (glucose target 5.0 mmol/L, insulin 150 mU x kg(-1) x h(-1)), and a hyperglycemic-hyperinsulinemic (glucose target 12.0 mmol/L, insulin 150 mU x kg(-1) x h(-1)) clamp on separate days, in random order. Skin microvascular flow was measured by laser Doppler flowmetry. Capillary permeability and density were determined by large-window sodium-fluorescein videodensitometry. Increases in serum soluble intercellular adhesion molecule-1 (sICAM-1) and plasma von Willebrand factor antigen (vWF-Ag) were considered to represent abnormal endothelial function. Hyperglycemia (P < 0.01) and hyperinsulinemia (P < 0.05) as well as both interventions combined (P < 0.001) induced an increase in laser Doppler flow, without capillary recruitment. Transcapillary leakage of sodium-fluorescein and sICAM-1 and vWF-Ag levels were unaffected by hyperglycemia or hyperinsulinemia. Microvascular permeability appears to be determined primarily by properties of the capillary wall and not by acute changes in local hemodynamics. The acute hyperglycemia- and hyperinsulinemia-induced vasodilatation is not accompanied by changes in microvascular permeability or endothelial markers.

Pathogenesis, clinical picture and treatment of von Willebrand's disease.

Von Willebrand's disease is probably the most common congenital bleeding disorder, with a prevalence close to 1% in some epidemiological studies. The disease is caused by a quantitative deficiency or a qualitative defect of the von Willebrand factor, which is a multimeric glycoprotein consisting of subunits of 2050 amino acids. The size of multimers ranges from approximately 500 kDa to 20 MDa. Each subunit consist of repeated domain structures. Several functional domains have been identified which can bind such structures as platelet receptors glycoprotein Ib or IIb/IIIA, heparin, collagen or factor VIII. The von Willebrand factor has two main functions in haemostasis, to promote normal platelet adhesion and to be a carrier protein for factor VIII. Von Willebrand's disease is divided into three major types and several subtypes depending on the quantity and quality of the von Willebrand factor in plasma and platelets. A new classification has recently been proposed. Typical symptoms are mucosal bleeding, easy bruising and increased bleeding tendency in connection with tooth extractions and other invasive procedures. Severe cases may have joint bleeding and other haemophilia-like bleeding. Desmopressin is the treatment of choice in mild cases, whereas more severe cases need treatment with factor VIII concentrates.

Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system.

An in vivo system of membranous bone formation during distraction has been investigated in order to follow cells that express vascular markers with the objective of understanding the neovascularization process. Concomitantly, sustained proliferation of preskeletal cells was achieved through the application of mechanical force. New capillaries and leading edges that arose by angiogenesis from the periosteal and mucosal surfaces and invaded the central zone of the regenerating distraction tissue temporally preceded the growth of delicate woven bone trabeculae from both edges of the cut bone. Concentrically arranged 'onion-like' configurations were abundant in paracentral zones and in association with mesenchymal condensations, suggesting their de novo formation in situ. Vascular specific markers, the angiopoietin receptor Tie-2 and factor VIII-related antigen (FVIIIrAg), were localized immunohistochemically in order to follow cells of vascular origin. Endothelial cells of the new capillaries, centrally located cells of the concentric configurations, pericytes, and most of the adjacent polygonal mesenchymal cells stained positively with specific antibodies to both antigens. Moreover, preosteoblasts and osteoblasts that lie adjacent to or already embedded in the osteiod of the newly formed trabeculae were also FVIIIrAg and Tie-2 immunopositive. As the source of the bone-forming cells in regenerating tissue during distraction is not yet fully understood, this observation might support the possibility of their vascular origin.