Assembly and Activation of Alternative Complement Components on Endothelial Cell-Anchored Ultra-Large Von Willebrand Factor Links Complement and Hemostasis-Thrombosis

Background

Vascular endothelial cells (ECs) express and release protein components of the complement pathways, as well as secreting and anchoring ultra-large von Willebrand factor (ULVWF) multimers in long string-like structures that initiate platelet adhesion during hemostasis and thrombosis. The alternative complement pathway (AP) is an important non-antibody-requiring host defense system. Thrombotic microangiopathies can be associated with defective regulation of the AP (atypical hemolytic-uremic syndrome) or with inadequate cleavage by ADAMTS-13 of ULVWF multimeric strings secreted by/anchored to ECs (thrombotic thrombocytopenic purpura). Our goal was to determine if EC-anchored ULVWF strings caused the assembly and activation of AP components, thereby linking two essential defense mechanisms.

Methodology/Principal Findings

We quantified gene expression of these complement components in cultured human umbilical vein endothelial cells (HUVECs) by real-time PCR: C3 and C5; complement factor (CF) B, CFD, CFP, CFH and CFI of the AP; and C4 of the classical and lectin (but not alternative) complement pathways. We used fluorescent microscopy, monospecific antibodies against complement components, fluorescent secondary antibodies, and the analysis of >150 images to quantify the attachment of HUVEC-released complement proteins to ULVWF strings secreted by, and anchored to, the HUVECs (under conditions of ADAMTS-13 inhibition). We found that HUVEC-released C4 did not attach to ULVWF strings, ruling out activation of the classical and lectin pathways by the strings. In contrast, C3, FB, FD, FP and C5, FH and FI attached to ULVWF strings in quantitative patterns consistent with assembly of the AP components into active complexes. This was verified when non-functional FB blocked the formation of AP C3 convertase complexes (C3bBb) on ULVWF strings.

Conclusions/Significance

AP components are assembled and activated on EC-secreted/anchored ULVWF multimeric strings. Our findings provide one possible molecular mechanism for clinical linkage between different types of thrombotic and complement-mediated disorders.     

Human Factor H-Related Protein 2 (CFHR2) Regulates Complement Activation

Mutations and deletions within the human CFHR gene cluster on chromosome 1 are associated with diseases, such as dense deposit disease, CFHR nephropathy or age-related macular degeneration. Resulting mutant CFHR proteins can affect complement regulation. Here we identify human CFHR2 as a novel alternative pathway complement regulator that inhibits the C3 alternative pathway convertase and terminal pathway assembly. CFHR2 is composed of four short consensus repeat domains (SCRs). Two CFHR2 molecules form a dimer through their N-terminal SCRs, and each of the two C-terminal ends can bind C3b. C3b bound CFHR2 still allows C3 convertase formation but the CFHR2 bound convertases do not cleave the substrate C3. Interestingly CFHR2 hardly competes off factor H from C3b. Thus CFHR2 likely acts in concert with factor H, as CFHR2 inhibits convertases while simultaneously allowing factor H assisted degradation by factor I.     

Cobra Venom Contains a Protein of the CRISP Family

Amino acid sequences of several fragments of the 25k protein (molecular mass 24 953 Da) previously isolated from cobra Naja kaouthia(Kukhtina et al.,Bioorg. Khim., 2000, vol. 26, pp. 803–807) were determined. Their comparison with the primary structures of known proteins showed that the 25k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.     

Ancient Origin of Human Complement Factor H

We studied the evolutionary history of two homologous proteins of the human complement system, factor H (FH) and the α chain of the C4b binding protein (C4bpα), and included in this study the related proteins from the barred sand bass (P. nebulifer) and the nematode C. elegans. Phylogenetic trees inferred from individual short consensus repeats (SCRs) and divergence among repeats from different genes suggest that human FH has a much closer evolutionary relationship to putative complement components from P. nebulifer and C. elegans than does the C4bpα. This indicates that a member of the alternative pathway of the complement system (FH) has an ancient origin, while a homologous member of the classical pathway (C4bpα) appeared later in evolutionary history as a result of gene duplication. The ancient evolutionary position of FH is in agreement with the suggestion that the alternative pathway of the complement system is older than the classical pathway. Phylogenetic analysis also shows that the sand bass cofactor protein SBP1 and cofactor related protein SBCRP-1 have diverged very recently. Received: 1 December 1997 / Accepted: 3 June 1998     

The Effects of Cobra Venom Factor,an Inhibitor of the Complement System,on the Sequence of Morphological Events in the Rat Kidney in Experimental Pyelonephritis

Acute experimental pyelonephritis has been produced by a combination of mechanical ureteral obstruction and intravenous injection of E. coli (strain IMRU-54). The effects of administration of cobra venom factor, an inhibitor of the complement system, on the sequence of morphologic events in the kidneys have been studied by light and electron microscopy.Pronounced bacterial colonization and suppression of the infiltration of acute inflammatory cells into the kidney were present in the cobra venom factor treated rats on day 2. In these rats, in which the infiltration of polymorphonuclear leukocytes was inhibited, renal structural damage was significantly reduced. The findings appear to indicate that the polymorphonuclear leukocytes infiltrating into the kidney play some role in damaging the renal parenchymal tissue in the early phase of E. coli induced acute pyelonephritis in rats.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.     

Effect of C4 Depletion on the Utilization of the Terminal Components of Guinea-pig Complement by Endotoxin

ENDOTOXIN and antigen-antibody complexes (Ag-Ab) are potent inflammatory agents which induce marked pathophysiological effects in a number of animal species. Both these agents lead to increased vascular permeability, local accumulations of polymorphonuclear leucocytes and alterations in the coagulability of blood¹. There is mounting evidence that these similarities derive from endotoxins and Ag-Ab being activators of the complement (C) system, an important mediator of inflammation¹'². When endotoxins or preformed Ag-Ab are reacted with normal guinea-pig (GP) serum so as to induce equivalent consumption of whole haemolytic C, there are, however, striking differences in the consumption profiles of the individual C components³. Although interaction of Ag-Ab with GP serum results in the consumption of CI, C4 and C2 as well as C3 to C9, the interaction of endotoxin with GP serum leads to a marked depletion of C3 to C9 with little or no detectable loss of C1,C4 and C2 (ref. 3).     

Protective Effect of a New Hypothalamic Peptide Against Cobra Venom and Trauma-Induced Neuronal Injury

A study of separate and combined actions of cobra venom (CV) and a new hypothalamic proline-rich polypeptide (PRP) isolated from magnocellular cells (NPV and NSO) on intoxication-and trauma, induced neuronal injury (during 3-4 weeks after hemisection with and without PRP treatment) was carried out. The registration of background and evoked impulse activity flow, changes in spinal cord (SC) inter- and motoneurons, responding to flexor, extensor, and mixed nerve stimulation in both acute and chronic experimental neurodegeneration was performed. The facilitating effect of PRP on the abovementioned neurons was revealed. High doses of CV that evoked the neurodegenerative changes demonstrated an inhibitory effect. In this case PRP treatment both before and after intoxication restored electrical neuronal activity to baseline level and higher. These results are evidence of protective action of PRP. The low doses of CV induced a facilitating effect. The combination of CV and PRP displayed an additive facilitating effect; in a number of cases the repeated administration of CV led to decrease of significant PRP effect till baseline level (for example, the inhibition after primary response prior to secondary late discharge). Greater liability of the secondary early and late long-time discharges of poststimulus responses, differently expressed in various neuron types of SC to chemical influences is of interest. PRP-induced inhibition of the paroxysmal activity related with CV action is also very interesting. Morpho-functional experiments with SC injury demonstrated the abolition of difference in the background and evoked SC neuronal activity below the section and on intact symmetric side after daily PRP administration for 3 weeks. PRP hindered the scar formation and activated neuroglia proliferation; it promoted white matter element growth, hampered the degeneration of cellular elements, and protected against tissue stress. Our results favor the combined use of PRP and CV in clinical practice for the treatment of neurodegeneration of toxic and traumatic origin, as well as specific neurodegenerative diseases such as Alzheimer's.     

Monoamine Oxidases A and B as Components of a Membrane Complex

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane-bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high-speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A₂, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.     

P-I Snake Venom Metalloproteinase Is Able to Activate the Complement System by Direct Cleavage of Central Components of the Cascade

Background

Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results

Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion

We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.     

Viper and Cobra Venom Neutralization by Alginate Coated Multicomponent Polyvalent Antivenom Administered by the Oral Route

Background

Snake bite causes greater mortality than most of the other neglected tropical diseases. Snake antivenom, although effective in minimizing mortality in developed countries, is not equally so in developing countries due to its poor availability in remote snake infested areas as, and when, required. An alternative approach in this direction could be taken by making orally deliverable polyvalent antivenom formulation, preferably under a globally integrated strategy, for using it as a first aid during transit time from remote trauma sites to hospitals.

Methodology/Principal Findings

To address this problem, multiple components of polyvalent antivenom were entrapped in alginate. Structural analysis, scanning electron microscopy, entrapment efficiency, loading capacity, swelling study, in vitro pH sensitive release, acid digestion, mucoadhesive property and venom neutralization were studied in in vitro and in vivo models. Results showed that alginate retained its mucoadhesive, acid protective and pH sensitive swelling property after entrapping antivenom. After pH dependent release from alginate beads, antivenom (ASVS) significantly neutralized phospholipaseA₂ activity, hemolysis, lactate dehydrogenase activity and lethality of venom. In ex vivo mice intestinal preparation, ASVS was absorbed significantly through the intestine and it inhibited venom lethality which indicated that all the components of antivenom required for neutralization of venom lethality were retained despite absorption across the intestinal layer. Results from in vivo studies indicated that orally delivered ASVS can significantly neutralize venom effects, depicted by protection against lethality, decreased hemotoxicity and renal toxicity caused by russell viper venom.

Conclusions/Significance

Alginate was effective in entrapping all the structural components of ASVS, which on release and intestinal absorption effectively reconstituted the function of antivenom in neutralizing viper and cobra venom. Further research in this direction can strategize to counter such dilemma in snake bite management by promoting control release and oral antivenom rendered as a first aid.     

广西产眼镜蛇毒酸性磷脂酶A2的分离纯化及性质研究

目的　从广西产中华眼镜蛇毒 ( N aja naja atra)中分离了一种新的磷脂酶 A2 ( PLA2 ) ,并研究其性质。　方法　磷脂酶 A2 ( PLA2 )的分离纯化采用 CM5 2、CM-Sepharose CL-6 B离子交换柱和 SepharoseCL-6 B凝胶柱 ,磷脂酶 A2 ( PLA2 )的性质采用经典方法进行。　结果　分离后的磷脂酶 A2 经过聚丙烯酰胺凝胶电泳、SDS-聚丙烯酰胺凝胶电泳和 HPLC检测 ,其为单一组分。SDS-PAGE测定它的分子量为1 5 0 0 0± 1 0 0 0。等电聚焦测得它的等电点为 6 .3。它的最适温度为 5 5℃ ,最适 p H值为 8.5。氨基酸成分分析表明该酶由 1 2 6个氨基酸组成 ,以 Asp、Ala、Gly、Cys居多。Fe3 、Zn2 、EDTA对其酶活力起抑制作用 ;K 、Ca2 、去垢剂对其活力起促进作用。荧光光谱分析表明该酶的色氨酸残基、组氨酸残基可能位于分子表面。药理实验表明 :该酶具有抗胰蛋白酶作用、抗凝血作用、间接溶血作用以及对青蛙具有心脏毒性。　结论　广西产中华眼镜蛇毒磷脂酶 A2 与其它来源的 PLA2 同源性高 ,但性质不尽相同。     

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β₂-glycoprotein I and interferes with innate immune recognition.     

Pasteurella pneumotropica Evades the Human Complement System by Acquisition of the Complement Regulators Factor H and C4BP

Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections.     

Expression and Subcellular Targeting of Human Complement Factor C5a in Nicotiana species

We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T₀ transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.     

抗蛇毒血清滴眼治疗眼镜蛇毒致眼外伤

目的观察单价抗眼镜蛇毒血清溶液滴眼治疗中华眼镜蛇毒(Naja naja atra,Chinese cobra)不慎进人眼睛引起外伤中毒性急性角膜炎的临床效果。方法观察1992～2002年我院急诊救治眼镜蛇喷毒或加工蛇毒时不慎蛇毒进人眼睛引起外伤性急性角膜炎8例男性病人,从受伤到就诊时间最快15min,最慢50min。就诊后立即用生理盐水冲洗受伤眼睛,紧接着给予单价抗眼镜蛇毒血清溶液滴人患眼0．5h后,用氯霉素眼药水、可的松(或地塞米松)眼药水交替滴眼,直至痊愈。结果使用抗蛇毒血清滴眼后局部疼痛、异物感等症状在20min内得到缓解,3天内眼睑红肿、结膜充血等角膜炎症状消失。8例病人均治愈,未留有后遗症。结论：单价抗眼镜蛇毒血清滴眼治疗中华眼镜蛇毒致眼外伤是非常方便有效的方法,可能与其能迅速中和眼睛内残留的蛇毒有关。