A review on bradykinin-related peptides isolated from amphibian skin secretion.

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure-function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future.

Kinin generation from exogenous kininogens at the surface of retinoic acid-differentiated human neuroblastoma IMR-32 cells after stimulation with interferon-γ.

Bradykinin-related peptides, kinins, ubiquitously occur in the nervous system and together with other pro-inflammatory mediators contribute to pathological states of that tissue such as edema and chronic pain. In the current work we characterized the kinin-forming system of neuronal cells obtained by differentiation of human neuroblastoma cell line IMR-32 with retinoic acid. These cells were shown to concentrate exogenous kinin precursors, kininogens, on the surface, release kinins from kininogens and subsequently convert kinins to their des-Arg metabolites. Significantly higher amounts of kinins and des-Arg-kinins were produced after cell stimulation with interferon-γ, a potent pro-inflammatory mediator involved in many neurological disorders. The expression of the major tissue kininogenase (the human kallikrein 1) and the major cell membrane-bound kininase (the carboxypeptidase M) also increased after cell stimulation with interferon-γ, suggesting the involvement of these enzymes in the kinin production and degradation, respectively. Interferon-γ was also able to up-regulate the expression of two known subtypes of kinin receptors. On the protein level, the changes were only observed in the expression of the des-Arg-kinin-specific type 1 receptor which functions in the propagation of the inflammatory state. Taken together, these results suggest a novel way for local kinin and des-Arg-kinin generation in the nervous tissue during pathological states accompanied by interferon-γ release.

A fish bradykinin (Arg0, Trp5, Leu8-bradykinin) from the defensive skin secretion of the European edible frog, Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle.

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val(1), Thr(6))-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes-common predators of these frogs. Here, we report the presence of (Arg(0), Trp(5), Leu(8))-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg(0), Trp(5), Leu(8))-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg(0), Trp(5), Leu(8))-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2.

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys(18) and Cys(24) and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133-160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr(6))-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.

The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization.

In two-dimensional (2-D) culture systems, we have previously shown that cleaved two-chain high-molecular-weight kininogen (HKa) or its domain 5 induced apoptosis by disrupting urokinase plasminogen activator (uPA) receptor (uPAR)-integrin signal complex formation. In the present study, we used a three-dimensional (3-D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3-D system, HKa significantly inhibited tube and vacuole formation as low as 10 nM, which represents 1.5% of the physiological concentration of high-molecular-weigh kininogen (660 nM), without apparent apoptosis. However, HKa (300 nM) completely inhibited tube formation and increased apoptotic cells about 2-fold by 20-24 h of incubation. uPA-dependent ERK activation and uPAR internalization regulate cell survival and migration. In a 2-D system, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In a 3-D system, we found that not only uPA-uPAR association but also the activation of ERK were inhibited by HKa. HKa disrupts the uPA-uPAR complex, inhibiting the signaling pathways, and also inhibits uPAR internalization and regeneration to the cell surface, thereby interfering with uPAR-mediated cell migration, proliferation, and survival. Thus, our data suggest that the suppression of ERK activation and uPAR internalization by HKa contributes to the inhibition of tube formation. We conclude that in this 3-D collagen-fibrinogen gel, HKa modulates the multiple functions of uPAR in endothelial cell tube formation, a process that is closely related to in vivo angiogenesis.

Elevated serum kininogen in patients with Paget's disease of bone: a role in marrow stromal/preosteoblast cell proliferation.

Paget's disease (PD) of bone is a chronic focal skeletal disorder characterized by excessive bone resorption followed by abundant new bone formation. Enhanced levels of IL-6, RANKL, M-CSF, and endothelin-1 have been associated with PD. In the present study, we identified increased serum levels (2 to 5-fold) of inflammatory cytokine, kininogen (KNG) in patients with PD compared to normal subjects. Treatment of pagetic bone marrow derived stromal/preosteoblast cells with recombinant KNG (25 ng/ml) for 24 h period resulted in a 5-fold increase in the levels of phospho-HSP27 and a 3-fold increase in ERK1/2 phosphorylation in these cells. However, pagetic stromal cells stimulated with KNG in the presence of ERK activation inhibitor peptide did not significantly affect the levels of phospho-HSP27. KNG increased normal and pagetic marrow stromal cell proliferation at 1.4-fold and 2.5-fold, respectively. KNG in the presence of an ERK inhibitor peptide did not stimulate pagetic marrow stromal cell proliferation. Furthermore, siRNA suppression of HSP27 expression significantly decreased KNG inhibition of etoposide-induced caspase-3 activation and apoptosis in these cells. In summary, KNG modulate bone marrow derived stromal/preosteoblast cell proliferation and suppress etoposide-induced apoptosis through ERK and HSP27 activation, respectively. These results implicate a pathophysiologic role for KNG in patients with PD.

T-kininogen can either induce or inhibit proliferation in Balb/c 3T3 fibroblasts, depending on the route of administration.

T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).

High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom.

High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.

Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production.

Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P(1)-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties.

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.

T-kininogen inhibits kinin-mediated activation of ERK in endothelial cells.

Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo.

A novel antithrombotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor-1 function.

The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.

The role of kinin B1 in the plasma extravasation of carrageenin-induced pleurisy.

The role of des-Arg9-bradykinin (des-Arg9-BK) and kinin B1 receptor in the plasma extravasation of rat carrageenin-induced pleurisy was investigated employing B1 receptor agonist and antagonists and kininogen-deficient rats. Expression of the B1 receptor mRNA in pleura was induced from 3 to 5 h after the injection of carrageenin into the pleural cavity of Sprague-Dawley rats. Exogenous injection of des-Arg9-BK into the pleural cavity provoked a significant increase in plasma extravasation in 5 h carrageenin-induced pleurisy, but not in 20 min kaolin-induced pleurisy. The level of immunoreactive des-Arg9-BK in the exudate of 5 h carrageenin-induced pleurisy was higher than that of bradykinin (BK). Administration of the B1 receptor antagonists, des-Arg9-[Leu8]-BK or des-Arg9-D-Arg-[Hyp3, Thi5, D-Tic7,Oic8]-BK significantly reduced the exudation rate. However, intrapleural administration of des-Arg9-BK to plasma kininogen-deficient. Brown Norway-Katholiek rats did not result in a further increase in the plasma extravasation. In conclusion, endogenously generated des-Arg9-BK could contribute to the plasma extravasation in carrageenin-induced pleurisy via mediation of the inducible B1 receptor.

Inhibition of vitronectin-mediated haptotaxis and haptoinvasion of MG-63 cells by domain 5 (D5(H)) of human high-molecular-weight kininogen and identification of a minimal amino acid sequence.

We found that human kinin-free high-molecular-weight kininogen (kf-HK) significantly inhibited vitronectin-mediated migration (haptotaxis) and invasive potentiation (haptoinvasion) of osteosarcoma (MG-63) cells but that HK, LK, the common heavy chain of HK and LK, and the light chain (D6(H)) of HK had no inhibitory effect. Recombinant GST-D5(H) (histidine-rich region of HK) obtained from Escherichia coli. (BL21) also inhibited both haptotaxis and haptoinvasion to about 30% of the control level in a dose-dependent manner. These findings suggest that a specific region of D5(H) is responsible for the inhibition of cell haptotaxis and haptoinvasion. Among the seven synthetic peptides covering D5(H), peptide H(479)KHGHGHGKHKNKGK(493) (P-5) inhibited both haptotaxis and haptoinvasion in a dose-dependent manner, suggesting that P-5 could possibly be utilized to prevent primary and secondary metastases of tumor cells.

Activation of the kinin-forming cascade on the surface of endothelial cells.

Activation of the plasma kallikrein-kinin forming cascade takes place upon incubation with human umbilical vein endothelial cells. The mechanism by which initiation occurs is uncertain. Zinc-dependent binding of plasma proteins to gC1qR, cytokeratin 1, and perhaps u-PAR is requisite for activation to take place. We demonstrate here that during a 2 hour incubation time plasma deficient in either factor XII or high molecular weight kininogen (HK) fails to activate, as compared to normal plasma, but with more prolonged incubation, factor XII-deficient plasma gradually activates while HK-deficient plasma does not. Our data support both factor XII-dependent (rapid) and factor XII-independent (slow) mechanisms; the latter may require a cell-derived protease to activate prekallikrein and the presence of zinc ions and HK.

Depressed modulation of oxygen consumption by endogenous nitric oxide in cardiac muscle from diabetic dogs.

Our previous study indicated that nitric oxide (NO)-dependent coronary vasodilation was impaired in conscious dogs with diabetes. Our goal was to determine whether modulation of O(2) consumption by NO is depressed in canine cardiac muscle after diabetes. Diabetes was induced by injection of alloxan (40-60 mg/kg iv), dogs were killed after diabetes was induced (4-5 wk), and the cardiac muscle from the left ventricle was cut into 15- to 30-mg slices. O(2) uptake by the muscle slices was measured polarographically with a Clark-type O(2) electrode. S-nitroso-N-acetylpenicillamine decreased O(2) consumption in normal and diabetic tissues (10(-4) M, 61 +/- 7 vs. 61 +/- 8%, P > 0.05). Bradykinin (10(-4) M)- or carbachol (CCh, 10(-4) M)-induced inhibition of O(2) consumption was impaired in diabetic tissues (51 +/- 6 vs. 17 +/- 4% or 48 +/- 4 vs. 19 +/- 3%, respectively, both P < 0.05 compared with normal). The inhibition of O(2) consumption by kininogen or kallikrein was depressed in diabetic tissues as well. In coronary microvessels from diabetic dogs, bradykinin or ACh (10(-5) M) caused smaller increases in NO production than those from normal dogs. Our results indicate that the modulation of O(2) consumption by endogenous, but not exogenous, NO is depressed in cardiac muscle from diabetic dogs, most likely because of decreased release of NO from the vascular endothelium.

Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1.

High molecular weight kininogen (HK) and its cleaved form (HKa) have been shown to bind to neutrophils. Based on studies using monoclonal antibodies (mAbs), we postulated that CD11b/CD18 (Mac-1) might be the receptor on the neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and Mac-1 had not been demonstrated. We therefore transfected HEK 293 cells with human Mac-1. Cell binding assays using fluorescein isothiocyanate-labeled HKa showed increased binding to the Mac-1 transfected cells compared with the control transfected cells. The binding was specific because unlabeled HKa, Mac-1-specific antibody, and fibrinogen can inhibit the binding of biotin-HKa to Mac-1 transfected cells. HKa bound to Mac-1 transfected cells (20 000 molecules/cell) with a K(d) = 62 nmol/L. To demonstrate directly the formation of a complex between HKa and Mac-1, we examined the interaction of HKa and purified Mac-1 in a cell-free system using an IAsys resonant mirror optical biosensor. The association and dissociation rate constants (k(on) and k(off), respectively) were determined, and they yielded a dissociation constant (K(d)) of 3.2x10(-9) mol/L. The functional significance of direct interaction of HKa to Mac-1 was investigated by examining the effect of HKa on cellular adhesion to fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in the injured vessel wall. HKa blocked the adhesion of Mac-1 transfected cells to fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt Mac-1-mediated cell-extracellular matrix and cell-cell adhesive interactions and may therefore influence the recruitment of circulating neutrophils/monocytes to sites of vessel injury. (Blood. 2000;95:3788-3795)

High molecular weight kininogen inhibition of endothelial cell function on biomaterials.

Synthetic vascular grafts implanted into humans fail to develop a complete endothelial lining. In previous studies, we have shown that high-molecular-weight kininogens (HMWK) adsorb to the surfaces of biomaterials. In addition, it has been demonstrated that these proteins modulate cellular function. In the present study, we report on the adhesion and proliferation of human umbilical-vein endothelial cells (HUVEC) on tissue culture polystyrene, glass, polyurethane, and Mylar(trade mark) surfaces coated with human HMWK, either single-chain HMWK (SC-HMWK) or double-chain HMWK (DC-HMWK). Surfaces coated with fibronectin served as a positive control for these experiments. Parallel experiments were performed in which HUVEC were allowed to migrate from crosslinked dextran microcarrier beads (Cytodex 2) onto HMWK-coated surfaces. Our results indicate that HMWK-coated surfaces inhibit endothelial cell adhesion, proliferation, and migration at 24 and 72 h, and this inhibition is concentration dependent. To determine a potential mechanism for this inhibitory phenomenon, cells were stained for cytoskeletal actin filaments using rhodamine-phalloidin. Endothelial cells on HMWK-coated surfaces displayed F-actin filament reorganization/disassembly, characterized by the absence of peripheral actin bands in focal adhesion contacts. We conclude that HMWK inhibit endothelial cell adhesion, proliferation, and migration on a variety of biomaterial surfaces. This inhibitory effect may play a role in promoting the lack of endothelialization in synthetic vascular grafts, which is thought to play a significant role in the failure of these devices.

Neutrophil adhesion on polyurethanes preadsorbed with high molecular weight kininogen.

Interaction of biomaterials with blood components including neutrophils is responsible for some of the clinical complications that have occurred in cardiopulmonary bypass, hemodialysis, and ventricular assist procedures. The possibility of inhibiting the initial adhesion of neutrophils to biomaterials has been studied extensively, but the problem remains unsolved. In this study, we investigated the effect of HK adsorption on polyurethane, a widely used component of extracorporeal and intracorporeal devices. HK and HKa were allowed to adsorb on 4 different charged polyurethanes: noncharged (PU), cationic (NR(4)), anionic (SO(3)), and zwitterionic (GPC) polyurethanes. The effect of kininogen adsorption on neutrophil adhesion, the surface density of the adsorbed kininogen, and the exposure of HK domains 3 and 5 (D(3) and D(5H)), which are responsible for the binding of HK to the neutrophil integrin alpha(m)beta(2) or Mac-1, were examined. On PU, NR(4), and SO(3), kininogen adsorption reached 80% of monolayer coverage when 100 pmol/mL or higher concentration of protein solutions were used. The NR(4) surface adsorbed the most kininogen along with a high exposure of D(3) and D(5H). The availability of D(3) and D(5H) allowed neutrophils to bind to the surface via the Mac-1 receptor; thus, on the NR(4) surface, adsorbed kininogens lost their antiadhesive property, which resulted in a high degree of neutrophil adhesion. Increasing Mac-1 expression by exposure to fMLP increased the neutrophil adhesion on this surface. In contrast, exposure of D(3) and D(5H) on SO(3) was significantly less, because HK binds to anionic surfaces with similar protein sequences used for cell binding. This low binding site exposure preserved the antiadhesive property of HK. GPC was resistant to neutrophil adhesion even in the absence of adsorbed kininogens because of its phosphorylcholine moiety. Thus, both SO(3) coupled with kininogen (or kininogen peptides) and GPC have the potential to markedly reduce neutrophil adhesion to biomaterial devices.

Prevalence and heterogeneity of antiphosphatidylethanolamine antibodies in patients with recurrent early pregnancy losses.

OBJECTIVE: To describe the prevalence of antiphospholipid antibodies to both anionic and zwitterionic phospholipids in women with early recurrent pregnancy losses (RPLs). DESIGN: Retrospective data analysis. SETTING: Tokai University Hospital, Kanagawa, Japan. PATIENT(S): One hundred thirty-nine patients with unexplained early RPLs. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Enzyme-linked immunosorbent assays were used to measure autoantibodies to phosphatidylethanolamine, cardiolipin, and phosphatidylserine. RESULT(S): Twenty-eight (20.1%), 17 (12.2%), and 2 (1.4%) patients of the 139 total patients were positive for immunoglobulin (Ig) G, IgM, and IgA antiphosphatidylethanolamine antibodies, respectively. Because 3 patients had two isotypes, 44 (31.7%) of the patients were positive for antiphosphatidylethanolamine antibodies. Six patients (4.3%) and 1 patient (0.7%) were positive for IgG and IgM antiphosphatidylserine antibodies, respectively. Seven patients (5%) were positive for beta2-glycoprotein I-independent anticardiolipin IgG, and 1 patient was positive for beta2-glycoprotein I-dependent anticardiolipin IgG. Two patients (1.4%) had lupus anticoagulant. CONCLUSION(S): Our data show a statistically stronger association between RPLs and antiphosphatidylethanolamine antibodies than between RPLs and antibodies to anionic phospholipids for early gestational losses. Our data suggest that antiphosphatidylethanolamine antibodies may be a risk factor in patients with early RPLs.