Plasma High Molecular Weight Kininogen Research Articles

The effect of estrogen-containing birth control pills on the constituents of bradykinin expression in plasma

Hereditary angioedema with C1-inhibitor deficiency (HAE-C1INH) is a rare autosomal disorder presenting with recurrent angioedema. Estrogen-containing medications trigger angioedema in some patients, and conversely, progesterone may decrease attack frequency. The mechanism by which estrogen may exacerbate angioedema in HAE-C1INH is not well characterized. Our aim was to investigate the link between estrogen and bradykinin constituents to better understand the specific underlying triggers that may exacerbate angioedema in patients with HAE-C1INH. As estrogen is contraindicated for patients with HAE-C1INH, females without a history of angioedema were recruited to evaluate whether estrogen-containing oral contraceptive pills (OCPs) alter plasma protein levels of bradykinin, cleaved high-molecular-weight kininogen (cHK), and activated factor XII (FXIIa). Blood (plasma) was collected before initiation of OCP administration and 3 months thereafter. High-molecular-weight kininogen (HK) was measured by ELISA and FXIIa and cHK were analyzed by Western blot analysis. A total of 12 adult females without HAE-CINH (aged <40 years) had a median baseline plasma HK level of 33,976 ng/mL. After 3 months of OCP therapy, their median HK levelincreased to 38,202 ng/mL. With OCPs, there was also a significant increase in level of FXIIa protein (P <.01), as well asan increase in cHK protein level. This preliminary study, performed in females without HAE-C1INH, suggests that estrogen may exacerbateangioedema by increasing the production of cHK and FXIIa.

Prospective study of plasma high molecular weight kininogen and prekallikrein and incidence of coronary heart disease, ischemic stroke and heart failure

IntroductionHigh molecular weight kininogen (HK) and prekallikrein (PK) are proteins in the kallikrein/kinin system of the coagulation cascade. They play an important role in the contact activation system of the intrinsic coagulation pathway, renin-angiotensin activation, and inflammation. Hence these proteins have been posited to affect the occurrence of cardiovascular events and thus to be potential therapeutic targets. Previous case-control studies have provided inconsistent evidence for an association of HK and PK with cardiovascular disease. MethodsIn the prospective population-based Atherosclerosis Risk in Communities(ARIC) Study, we used Cox proportional hazards regression models to investigate the association in 4195 middle-aged adults of plasma HK and PK concentrations in 1993–95 (linearly and in quartiles) with incident coronary heart disease, ischemic stroke, and heart failure through 2016. ResultsOver a mean of 18 years follow-up, we identified incident cardiovascular events (coronary heart disease and ischemic stroke) in 618 participants and heart failure in 667. We observed no significant relation between HK or PK and cardiovascular disease or heart failure, before and after adjusting for several potential confounding variables. ConclusionsWe found no compelling evidence to support an association of plasma HK or PK concentrations with incident CHD, ischemic stroke, or heart failure.

An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema.

Hereditary angioedema (HAE) is a genetic disorder mostly caused by mutations in the C1 esterase inhibitor gene (C1INH) that results in poor control of contact pathway activation and excess bradykinin generation. Bradykinin increases vascular permeability and is ultimately responsible for the episodes of swelling characteristic of HAE. We hypothesized that the use of RNA interference (RNAi) to reduce plasma Factor XII (FXII), which initiates the contact pathway signaling cascade, would reduce contact pathway activation and prevent excessive bradykinin generation. A subcutaneously administered GalNAc-conjugated small-interfering RNA (siRNA) targeting F12 mRNA (ALN-F12) was developed, and potency was evaluated in mice, rats, and cynomolgus monkeys. The effect of FXII reduction by ALN-F12 administration was evaluated in two different vascular leakage mouse models. An ex vivo assay was developed to evaluate the correlation between human plasma FXII levels and high-molecular weight kininogen (HK) cleavage. A single subcutaneous dose of ALN-F12 led to potent, dose-dependent reduction of plasma FXII in mice, rats, and NHP. In cynomolgus monkeys, a single subcutaneous dose of ALN-F12 at 3 mg/kg resulted in >85% reduction of plasma FXII. Administration of ALN-F12 resulted in dose-dependent reduction of vascular permeability in two different mouse models of bradykinin-driven vascular leakage, demonstrating that RNAi-mediated reduction of FXII can potentially mitigate excess bradykinin stimulation. Lastly, ex vivo human plasma HK cleavage assay indicated FXII-dependent bradykinin generation. Together, these data suggest that RNAi-mediated knockdown of FXII by ALN-F12 is a potentially promising approach for the prophylactic treatment of HAE.

Limited role of kininogen in the host response during gram-negative pneumonia-derived sepsis.

High-molecular-weight kininogen (HK), together with factor XI, factor XII and prekallikrein, is part of the contact system that has proinflammatory, prothrombotic, and vasoactive properties. We hypothesized that HK plays a role in the host response during pneumonia-derived sepsis. To this end mice were depleted of kininogen (KNG) to plasma HK levels of 28% of normal by repeated treatment with a specific antisense oligonucleotide (KNG ASO) for 3 wk before infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways. Whereas plasma HK levels increased during infection in mice treated with a scrambled control ASO (Ctrl ASO), HK level in the KNG ASO-treated group remained reduced to 25-30% of that in the corresponding Ctrl ASO group both before and after infection. KNG depletion did not influence bacterial growth in lungs or dissemination to distant body sites. KNG depletion was associated with lower lung CXC chemokine and myeloperoxidase levels but did not impact neutrophil influx, lung pathology, activation of the vascular endothelium, activation of the coagulation system, or the extent of distant organ injury. These results were corroborated by studies in mice with a genetic deficiency of KNG, which were indistinguishable from wild-type mice during Klebsiella-induced sepsis. Both KNG depletion and KNG deficiency were associated with strongly reduced plasma prekallikrein levels, indicating the carrier function of HK for this zymogen. This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia-derived sepsis.

An essential role of high-molecular-weight kininogen in endotoxemia.

In this study, we show that mice lacking high-molecular-weight kininogen (HK) were resistant to lipopolysaccharide (LPS)-induced mortality and had significantly reduced circulating LPS levels. Replenishment of HK-deficient mice with human HK recovered the LPS levels and rendered the mice susceptible to LPS-induced mortality. Binding of HK to LPS occurred through the O-polysaccharide/core oligosaccharide, consistent with the ability to bind LPS from K. pneumoniae, P. aeruginosa, S. minnesota, and different E. coli strains. Binding of LPS induced plasma HK cleavage to the two-chain form (HKa, containing a heavy chain [HC] and a light chain [LC]) and bradykinin. Both HKa and the LC, but not the HC, could disaggregate LPS. The light chain bound LPS with high affinity (K d = 1.52 × 10-9 M) through a binding site in domain 5 (DHG15). A monoclonal antibody against D5 significantly reduced LPS-induced mortality and circulating LPS levels in wild-type mice. Thus, HK, as a major LPS carrier in circulation, plays an essential role in endotoxemia.

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn+2, HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn2+. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

The Role of Urokinase Receptor (uPAR) In Efferocytosis: uPAR Engulfs Apoptotic Cells via a Phosphatidylserine Pathway Mediated by Plasma High Molecular Weight Kininogen

AbstractAbstract 929Phagocytosis of apoptotic cells by phagocytes, also known as efferocytosis, is essential for maintaining normal tissue homeostasis and regulating immune responses. Defects in rapid clearance of apoptotic cells lead to the release of immunogenic cellular contents, which may cause tissue damage and autoimmune disease. Phagocytic receptors differentiate apoptotic cells from viable cells by recognizing ‘don't eat- or eat-me’ signals on the cell surface. Recently, we and others have reported the role of uPAR in mediating efferocytosis. In this study, we examined the mechanism by which uPAR recognizes and internalizes apoptotic cells. By flow cytometry-based in vivo and in vitro phagocytosis assay, we found that in knockout mice the lack of uPAR expression on macrophages decreased their apoptotic cell engulfing activity by >35%. Conversely, soluble uPAR and polyclonal anti-uPAR antibodies (Ab) suppressed the internalization of apoptotic cells by macrophages. However, there was no defect in uPAR-/- macrophage uptake of viable cells, suggesting that uPAR plays a specific role in phagocytosis of apoptotic cells. We established a HEK 293 cell line expressing human full-length uPAR (293-uPAR). In these cells, uPAR-mediated phagocytosis of apoptotic cells was completely blocked by annexin V in the presence of calcium. The effect of annexin V was not observed in the absence of calcium, indicating that uPAR internalizes apoptotic cells through a phosphatidylserine pathway. We also found that uPAR-mediated uptake of apoptotic cells was completely prevented under serum-free conditions. To identify plasma proteins that may opsonize the uPAR function, we used immunodepletion method to test three known uPAR-binding proteins, vitronectin, uPA and high molecular weight kininogen (HK). Depletion of HK from serum by a polyclonal anti-HK Ab significantly reduced the engulfment of apoptotic cells by either macrophages or 293-uPAR cells in a co-culture system. In contrast, depletion of vitronectin or uPA from serum had little effect. uPAR is a GPI-anchored protein. Upon sucrose gradient ultracentrifugation, the majority of uPAR molecules were co-localized with membrane-bound HK in lipid rafts. The binding capacity of HK to apoptotic cell surface was further analyzed by flow cytometry. Phycoerythrin-labeled HK bound to apoptotic cells in a concentration-dependent manner, saturating at 300 nM. In contrast, HK did not bind to viable cells at concentrations up to 1200 nM. It is known that HK is a key component of the plasma contact system and that apoptotic cells potentiate factor Xa formation. Our new findings of the uPAR-HK-phosphatidylserine axis in efferocytosis suggest that this pathway may modulate the coagulation cascade on the surface of apoptotic cells. This pathway may also play a role in the pathogenesis of autoimmune and thrombotic disease. Disclosures:No relevant conflicts of interest to declare.

Contact activation of kallikrein–kinin system by superparamagnetic iron oxide nanoparticles in vitro and in vivo

Previously we reported that plasma kallikrein and high molecular weight kininogen attach to the surface of dextran-coated superparamagnetic iron oxide nanoparticles (SPION) through the incompletely covered iron oxide core (Simberg et al., Biomaterials, 2009). Here we show that SPION also activate kallikrein–kinin system in vitro and in vivo. The serine protease activity of kallikrein was stably associated with SPION and could be detected on the nanoparticles even after extensive washing steps. The enzymatic activity was not detectable in kininogen-deficient and Factor XII-deficient plasma. The enzymatic activation could be blocked by precoating SPION with histidine-rich Domain 5 (D5) of kininogen. Importantly, the kallikrein activity was detectable in plasma of SPION-injected, but not of D5/SPION-injected mice. Tumor-targeted SPION when injected into kininogen-deficient and control mice, produced high levels of vascular clotting in tumors, suggesting that kallikrein activation is not responsible for the nanoparticle-induced thrombosis. These data could help in understanding the toxicity of nanomaterials and could be used in designing nanoparticles with controlled enzymatic activity.

Neutrophil‐derived heparin binding protein (HBP) is an endogenous activator of the kallikrein‐kinin system

Adhesion of polymorphonuclear leukocytes (PMN) to the endothelial cell (EC) lining leads to release of heparin binding protein (HBP) causing increased vascular permeability. We have previously reported data suggesting that the kallikrein‐kinin system (KKS) is involved in PMN‐evoked plasma extravasation. Plasma prekallikrein (PK), factor XII (FXII) and high molecular weight kininogen (HK) are principal components of the KKS. Activated PK cleaves HK, liberating bradykinin (BK) which triggers EC contraction. Endogenous activators of this system are poorly defined. Here, we investigated the role of HBP in the activation of the KKS. Stimulation of EC monolayers with HBP caused an increase in paracellular permeability that was abolished with BK B1 and B2 receptor antagonists or a peptide displacing HK from the EC surface. Western blot analysis further revealed that HBP is able to induce cleavage of HK on EC. Thioglycollate‐induced pleurisy in the mouse was used to study PMN‐evoked plasma changes in vascular permeability in vivo. Plasma extravasation was markedly increased 4 h after thioglycollate injection. Treatment with BK receptor antagonists or an antibody to HBP significantly reduced the plasma exudation. These results demonstrate a critical role of the KKS in PMN‐evoked increase in vascular permeability. The data suggest that PMN‐derived HBP may constitute an endogenous activator of this system.

Deletion of Murine Kininogen Gene 1 (mKNG1) Causes Loss of Plasma Kininogen and Delays Thrombosis.

High molecular weight kininogen (HK) plays an important role in the assembly of the plasma kallikrein-kinin system. While the human genome contains a single copy of the kininogen gene, three copies exist in the rat (one encoding K-kininogen and two encoding T-kininogen). Here, we confirm that the mouse genome contains two homologous kininogen genes, mKng1 and mKng2. These genes are located on chromosome 16 in a head to head orientation with ∼ 30 kB intervening sequence, and are expressed in a tissue-specific manner. To determine the roles of these genes in murine development and physiology, we disrupted mKng1, which is expressed primarily in the liver. mKng1−/ − mice were viable, and immunoblotting using anti-bradykinin antibodies indicated a marked reduction in plasma HK and low molecular weight kininogen (LK), as well as ΔmHK-D5, a novel kininogen isoform that lacks kininogen domain 5. Clotting studies were also consistent with marked HK deficiency. Moreover, despite normal tail vein bleeding times, mKng1−/− mice displayed a significantly prolonged time to carotid artery occlusion following Rose Bengal administration and laser induced arterial injury. These results suggest that a single gene, mKng1, is responsible for production of plasma kininogen, and that plasma high molecular weight kininogen may contribute to induced arterial thrombosis in mice.

The Fragments of Bovine High Molecular Weight Kininogen Promote Osteoblast Proliferation In Vitro

High molecular weight (HMW) kininogen is known to be a large plasma protein and cleaved by plasma proteinase kallikrein, then it generates four fragments in the blood coagulation cascade: heavy chain, bradykinin, fragment 1.2, and light chain. The fragment 1.2 has also been found in the basic protein fraction of bovine milk as a bioactive protein which promotes osteoblast proliferation. The milk basic protein has been shown to be a multi functional edible protein which promotes bone formation and inhibits bone resorption. In the present study, we purified the fragment 1.2 from bovine plasma and assessed it could promote osteoblast proliferation and posses the activity after pepsin digestion. Purified plasma HMW kininogen did not promote the proliferation, however, the kallikrein-cleaved HMW kininogen promoted the proliferation. The fragment 1.2, purified from the proteolysate, also promoted the proliferation. The pepsin digestion was performed according to the method of the assessment of allergenesity of genetically modified crops. After pepsin digestion, the fragment 1.2 generated resistant fragments and showed the promoting activity of osteoblast proliferation. These results suggest that the enzymatically-digested fragments of bovine HMW kininogen are able to be a naturally occurred active protein that promotes the bone formation by oral administration.

High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin-1β through the NFκ B, JNK and p38 MAP Kinase Signaling Pathways in Monocytes.

Plasma high molecular weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to cleaved HK (HKa). We have reported that HKa releases cytokines (TNFα , IL-1β , IL-6) and chemokines (IL-8 and MCP-1) from isolated human peripheral blood monocytes. At a concentration of 600nM, Glutathione -S- transferase (GST) fusion proteins of kininogen domain 3 (D3), E7P - an active fragment of domain 3 (aaG255-Q292), HK domain 5 (D5), and D5 recombinant peptide HG (aa K420-D474) each stimulated secretion of IL-1β from monocytes. Receptors on monocytes including Mac-1, LFA-1, uPAR and gC1qR are required for IL-1β secretion from monocytes. We now report the signaling pathways and evidence for synthesis of IL-1 β in monocytes stimulated by HKa. Inhibitors of signaling pathways initiated by NFkB, JNK and p38, but not ERK, decreased IL-1b release from monocytes. A specific inhibitor of NFkB, MG-132 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 69.7, 67.3, 88.5% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 83.4% by MG-132 (100 μM). A specific inhibitor of JNK, SP 600125 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 55.7, 76.3, 78.9% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 90.23% by SP 600125 (100 μM). A specific inhibitor of p38, SB202190 (1,10 and 100 μM) significantly reduced IL-1β release from monocytes by 27.8, 14.2, 91.0% respectively, when stimulated by E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 76.8 % by SB202190 (100 μM). In contrast, the ERK activation inhibitor U0126 (1, 10 and 100 μM) failed to inhibit GST-E7P-induced release of IL-1β from monocytes but the release of IL-1β with LPS (10 μg/ml), was blocked by 77.4% at 100 μM. After monocytes (4 X 106/ml) were treated with HKa (600 nM) or LPS (10 ng/ml), total RNA was extracted and RT-PCR reactions for expression of IL-1β and gC1qR mRNA using specific primers were carried out. PCR products was separated in 4% ethidium bromide-stained agarose gels and photographed. No IL-1b mRNA was detected prior to exposure to HKa or LPS but both were detected at 1 hour. In contrast, gC1qR mRNA was present without stimulation by HKa. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by stimulating the synthesis and release of IL-1β from human monocytes using intracellular signaling pathways initiated by uPAR, β 2 integrins and gC1qR receptors.

Increased activity of plasma and tissue kallikreins, plasma kininase II and salivary kallikrein in pemphigus foliaceus (fogo selvagem)

Pemphigus foliaceus (PF) is an autoimmune blistering disease of unknown aetiology, which is endemic in Brazil. Although the pathogenesis of PF is still unknown, proteins of the contact system have been implicated. As the components of the kinin system may interact with those of the contact system, in this study we evaluated the plasma levels of high-molecular-weight kininogen (HK) and low-molecular-weight kininogen (LK), and the activity of plasma kallikrein, tissue kallikrein and kininase II in plasma of patients with PF presenting with Nikolsky's sign. As kidneys and salivary glands are relevant sources of tissue kallikrein for plasma, we also evaluated urinary/salivary kallikrein and urinary kininase II activities. Fifteen patients and 15 age- and sex-matched controls were studied. Kininogen levels were determined by enzyme-linked immunosorbent assay, and the activities of kallikreins and kininase II were determined using selective chromogenic substrates. Compared with controls, plasma HK levels were decreased (P = 0.031), whereas the activities of plasma kallikrein, tissue kallikrein and kininase II in plasma, and the activity of salivary kallikrein, were increased in patients (P < 0.001 for each comparison). Plasma levels of LK and the activities of urinary kallikrein and urinary kininase II were not significantly different from controls. Diminished levels of HK associated with increased activities of plasma kallikrein and kininase II indicate that the kinin system is activated at the systemic level in PF. As active plasma kallikreins may act on some proteins of the contact system, it is possible that the enzyme may contribute to blister formation. The further observation of an increased tissue kallikrein activity at the systemic and saliva levels may be interpreted as a systemic reflex of skin inflammation. Whether the activation of the kinin system is a cause or a consequence of blister formation needs further clarification.

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation

AbstractCrohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.

Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis

Little is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 microg/ml [range 22-99 microg/ml]) and LK (58 microg/ml [15-100 microg/ml]) than normal subjects (HK 83 microg/ml [65-115 microg/ml]; LK 80 microg/ml [45-120 microg/ml]) (p<0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P<0.0001) and albumin (P<0.0001 and P<0.001) and inversely to the Child-Pugh score (P<0.0001) and to prothrombin time ratio (P<0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

Biologic Activities of the Contact Factors In Vivo

Three plasma proteins, namely, Factor XII (Hageman factor), prekallikrein (Fletcher factor), and high molecular weight kininogen (Williams-Fitzgerald-Flaujeac factor), have been shown to be required for the activation of the surface-mediated pathway, or “contact system.” In vitro, such as in the activated partial thromboplastin test or on exposure to artificial surfaces, factor XII (FXII) is autoactivated to factor XIIa, which cleaves plasma prekallikrein (PK) by limited proteolysis into the active enzyme, plasma kallikrein (Fig. 1). In contrast, in vivo, high molecular weight kininogen (HK) binds to the endothelial cell surface associated with PK in a noncovalent complex. PK is activated by an endothelial cell cysteine protease to kallikrein which cleaves prourokinase to urokinase and factor XII to factor XIIa (Fig. 2). Thus, on cells, PK activation initiates the contact system, while on artificial surfaces, FXII is the initiating molecule. HK is also the substrate of plasma kallikrein, which cleaves to yield kinin-free HK (HKa) and bradykinin (Fig. 1). Although the genetically determined deficiencies of FXII, PK, and HK display abnormal in vitro plasma coagulation, none of these deficiencies is associated with a hemorrhagic diathesis. However, Factor XII, PK and HK display a wide variety of important interactions with other plasma proteins. In vitro, activated factor XII activates factor XI, factor VII and the C1 component of the complement system. Plasma kallikrein activates prourokinase (Fig. 1), prorenin, and factor XII, and cleaves HK, releasing bradykinin. Each contact protein interacts with blood and vascular cells. Factor XII downregulates a monocyte Fc receptor, releases IL-1 and IL-6 from monocytes and macrophages, and stimulates neutrophils, Plasma kallikrein activates neutrophils and potentiates fibrinolysis on endothelial cells. HK inhibits adhesion of neutrophils to artificial surfaces, enhances cellular fibrinolysis, and inhibits thrombin-induced platelet activation. Knowledge of these biochemical and cellular reactions has led to an understanding of the role of contact proteins in inflammation, fibrinolysis, cell adhesion, platelet activation and angiogenesis, and an appreciation of their participation in various pathophysiologic conditions. The molecular biology, structure and biochemical properties of FXII, PK and HK have recently been comprehensively reviewed. This review will focus on their participation in vivo in pathophysiologic processes including hypotension, inflammation, fibrinolysis, cell adhesion, angiogenesis and thrombosis.

The Pharmacokinetics of the Kininogens

Recent evidence has shown that plasma high molecular weight kininogen and both kininogens have the ability to modulate prekallikrein activation and thrombin-induced platelet activation, respectively. However, nothing is known about the plasma clearance and tissue distribution of these proteins. We examined the in vivo pharmacokinetics of high (HK) and low (LK) molecular weight kininogens in rats. 125I-HK and -LK molecular weight kininogens’ clearance in rats best-fitted a biexponential model. For HK, the t 1/2α and t 1/2β were 0.6 and 9.5 h and for LK, 0.78 and 7.4 h, respectively. 125I-kinin-free HK (cleaved HK) was cleared with a t 1/2α and t 1/2β of 0.45 and 9.9 h, respectively. 125I-Domain 3 of kininogens was cleared with a t 1/2β and t 1/2c of 0.99 and 13.3 h, respectively. HK was mostly concentrated in lung; LK, domain 3, and cleaved HK were mostly concentrated in kidney. The kininogens were also concentrated in liver, spleen, and skin. These studies indicate that protein size rather than form is the major determinant of its clearance. Furthermore, the distribution of the kininogens is where bradykinin metabolism and activity are well described.

Bradykinin release from high molecular weight kininogen and increase in plasma kallikrein-like activity following sensory stimulation by food in the rat.

Cephalic stimulation by food elicits, among other responses, dilatation of mesenteric blood vessels preparatory for digestion. The possible participation of bradykinin (BK), a powerful endogenous vasodilator, in this response was studied in fasted rats prior and following stimulation by sight and scent of food (sensory stimulation, SS), actual ingestion being denied to the animals. BK content of plasma high (HK) and low molecular weight kininogen (LK) was determined by bioassay on the atropinized, antihistamine-treated isolated guinea-pig ileum following release by trypsin from heat/acid denatured plasma. BK corresponding to LK was estimated in plasma which prior to denaturation had been incubated with kaolin, a process which leads to quantitative release and inactivation of BK from HK, but does not affect LK. BK corresponding to (HK + LK) was determined in plasma not exposed to kaolin. BK contained in HK was the difference between BK of (HK + LK) and of BK of LK. Plasma and glandular kallikreins were estimated by fluorimetry, using specific synthetic substrates. A 40.6+/-4.0% decrease (P<0.001) of BK in HK occurred in rats after 90 s of SS; LK remained unaffected. Ten minutes of SS did not result in further change. Atropine inhibited the effect of SS. Return of HK to pretreatment levels occurred when, following 90s of SS, rats were allowed to rest for 60 min in the absence of food. Renewed capacity to respond to SS was then observed. Plasma kallikrein, but not glandular kallikrein, increased in plasma of rats after SS. Increased free BK was detected in the circulation of Enalapril-protected rats after SS. Electrical stimulation of the distal sector of the sectioned left abdominal vagus nerve of Nembutal-anesthetized fasted rats reproduced the effect of SS on HK. It is concluded that visuo-olfactory stimulation by food generates nerve impulses, possibly carried by the vagus nerve, which by activating plasma kallikrein lead to cleavage of circulatory HK and release of BK in the rat.

Gamma Interferon Administration to Patients with Atopic Dermatitis Inhibits Fibrinolysis and Elevates C1 Inhibitor

Recombinant human gamma interferon was used to treat 10 atopic dermatitis patients. Recombinant gamma interferon was administered weekly for three consecutive days at 50 μg/M 2 SQ for four weeks. All patients’ dermatitis improved with recombinant gamma interferon therapy and plasma tumor necrosis factor-α levels rose with treatment. Recombinant gamma interferon treatment positively correlated with reduced total plasma fibrinolysis as measured by the fibrin lysis plate, plasmin-α 2antiplasmin complexes, and tissue type plasminogen activator levels. Accordingly, plasminogen activator inhibitor levels increased. Treatment also was associated with a transient increase in thrombin-antithrombin III complexes. Recombinant gamma interferon resulted in a significant increase in C1 inhibitor antigen but not activity. Plasma prekallikrein, high molecular weight kininogen, and factor XII levels were not decreased. However, 5 of the 10 atopic dermatitis patients before therapy had circulating cleaved plasma high molecular weight kininogen detected on immunoblot, indicating prior kallikrein formation. The cleaved, circulating plasma high molecular weight kininogen disappeared in four out of the five original patients who were reexamined at one year after treatment. These combined data indicated that recombinant gamma interferon treatment reduced total plasma fibrinolysis. In untreated atopic dermatitis, circulating cleaved high molecular weight kininogen also may be a presenting manifestation.