Mapping the interaction between high molecular mass kininogen and the urokinase plasminogen activator receptor

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase.     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.     

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.     

Inhibition of platelet adhesion and aggregation by a defined region (Gly-486-Lys-502) of high molecular weight kininogen

Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind two-chain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the alpha(IIb)beta(3) integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486-Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-alpha(IIb)beta(3)-integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486-Lys-502 abrogated the alpha(IIb)beta(3)-integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486-Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new anti-thrombotic compounds may become available on the basis of peptide Gly-486-Lys-502 of HK domain 5.     

Kinetic analysis of the role of zinc in the interaction of domain 5 of high-molecular weight kininogen (HK) with heparin

Previous investigations have shown that HK and its light chain bind heparin, preventing the enhancement of antithrombin inhibition of thrombin and potentiating the inhibition of plasma kallikrein by antithrombin. We found that both HK and HKa bound heparin, but HK exhibited a greater affinity. We therefore localized the binding sites for heparin on HK. HK domains 5 and 6 of the light chain as well as domain 3 from the heavy chain, expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli, were tested for binding to immobilized heparin by surface plasmon resonance using a BiaCore 2000 instrument. GST-D5, but not GST-D3, GST-D6, or GST, bound to heparin when the recombinant domains were present at a concentration of 70 nM. To localize more precisely the amino acid sequences on D5, both of the subdomains, histidine-glycine-rich GST-(K420-D474) and histidine-glycine-lysine-rich GST-(H475-S626), were expressed and tested for binding to immobilized heparin. The K(d) was much lower for GST-(K420-D474) than for GST-(H475-S626) in the presence or absence of Zn(2+). GST-(K420-D474) was effective in decreasing the rate of inactivation of thrombin by antithrombin in the presence of heparin and Zn(2+), while GST-(H475-S626) had no effect. We conclude that the binding of heparin to HK is a complex function of Zn(2+) interacting with histidines in the sequence K420-D474 to create high-affinity binding sites. HK has the potential to be an important modulator of heparin therapy.     

Conformation of high molecular weight kininogen: effects of kallikrein and factor XIa cleavage

The effect of kallikrein and factor XIa proteolysis of high molecular weight kininogen (HK) was investigated. Circular dichroism (CD) spectroscopy showed that cleavage of HK by plasma kallikrein or urinary kallikrein, both of which result in an active cofactor (HKa), results in conformational change that is characterized by increase in CD ellipticity at 222 nm. This suggests an increase in organized secondary structures. By contrast, cleavage of HK by factor XIa which results in an inactive cofactor (HKi) is characterized by a dramatic decrease in CD ellipticity at 222 nm suggesting an entirely different type of conformational change. The intrinsic fluorescence of HK is enhanced after cleavage by all three proteases. These conformational changes may play a role in determining the structure and function of HKa and HKi.     

Ferritin blocks inhibitory effects of two-chain high molecular weight kininogen (HKa) on adhesion and survival signaling in endothelial cells

Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with α5β1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa.     

Apoptotic effect of cleaved high molecular weight kininogen is regulated by extracellular matrix proteins

We previously reported that cleaved high molecular weight kininogen (HKa) and its domain 5 (D5) inhibit critical steps required for angiogenesis and in vivo neovascularization (Colman et al. 2000: Blood 95:543-550). We have further shown that D5 is able to induce apoptosis of endothelial cells, which may represent a critical part of the anti-angiogenic activity of HKa and D5 (Guo et al. 2001: Arterioscler Thromb Vasc Biol 21:1427-1433). In this study, we demonstrate that HKa- and D5-induced apoptosis is closely correlated with their anti-adhesive effect. An important new finding is that the apoptotic activity of HKa and D5 is highly regulated by their interactions with different extracellular matrix (ECM) proteins. HKa inhibited cell adhesion to vitronectin (Vn, 90%) and gelatin (Gel) (40%), but it had no apparent effect on cell adhesion to fibronectin (Fn). D5 showed a similar pattern on cell adhesion but was less potent than HKa. HKa induced apoptosis of endothelial cells grown on Vn and Gel but not cells grown on Fn which closely parallels with its anti-adhesive potency. Further results revealed that the anti-adhesive effect and the apoptotic effect of HKa are associated with its ability to inhibit phosphorylation of focal adhesion kinase (FAK) and paxillin, two important signal molecules required for cell adhesion and cell viability. We conclude that the anti-adhesive activity of HKa and D5 is responsible for their apoptotic effect and that Vn is likely an ECM component that mediates the effect of HKa and D5.     

Antithrombotic activity of kininogen is mediated by inhibitory effects of domain 3 during arterial injury in vivo

High-molecular-weight kininogen (HK) and its domain 3 (D3) exhibit anticoagulant properties and inhibit platelet activation at low thrombin concentration in vitro. We hypothesized that the rapid occlusive thrombosis in HK-deficient (HKd) rats following endothelial injury of the aorta results from enhanced platelet aggregation by thrombin. The effects of D3 (G235-M357) or D3-derived peptides on thrombosis in vivo were tested. D3 and its exon 7C terminal peptide (E7CP, K270-Q292), expressed as glutathione S-transferase (GST) fusion proteins (GST-D3, GST-E7CP), or GST alone, as well as cleaved HK (HKa) or synthetic peptide E7CP, were infused intravenously 10 min before endothelial injury. Blood flow was reduced down to 10% of baseline flow within 28 +/- 5.2 min by a platelet-fibrin thrombus in GST-treated HKd rats compared with >240 min in GST-treated normal HK rats (wild type). GST-D3, GST-E7CP, HKa, or E7CP infusion prolonged the flow time to 233, >240, 223, and >240 min, respectively, in HKd rats. When GST-E7CP was infused 10 min after the injury, blood flow was maintained for >240 min. Thrombin-antithrombin concentrations were elevated by injury in HKd rats receiving GST from 35 to 55 microg/l and decreased with GST-E7CP, HKa, or E7CP reconstitution to 40, 15, and 9 microg/l, respectively. We conclude that HKd rats are prothrombotic and that HKa, kininogen D3, and its fragment E7CP modulate arterial thrombosis after endothelial injury.     

Local bradykinin formation is controlled by glycosaminoglycans

Bradykinin is a potent inflammatory mediator that induces vasodilation, vascular leakage, and pain sensations. This short-lived peptide hormone is liberated from its large precursor protein high molecular weight kininogen (HK) through the contact system cascade involving coagulation factor XII and plasma kallikrein. Although bradykinin release is well established in vitro, the factors and mechanisms controlling bradykinin generation in vivo are still incompletely understood. In this study we demonstrate that binding of HK to glycosaminoglycans (GAGs) of the heparan and chondroitin sulfate type efficiently interferes with bradykinin release in plasma and on endothelial surfaces. Proteolytic bradykinin production on endothelial cells is restored following degradation of cell surface GAG through heparinase. Alternatively, application of HK fragments D3 or light chain, which compete with uncleaved HK for cell binding, promote kininogen proteolysis and bradykinin release. Intravital microscopy revealed that HK fragments increase bradykinin-mediated mesentery microvascular leakage. Topical application of D3 or light chain enhanced bradykinin generation and edema formation in the mouse skin. Our results demonstrate that bradykinin formation is controlled by HK binding to and detachment from GAGs. Separation of the precursor from cell surfaces is a prerequisite for its efficient proteolytic processing. By this means, fragments arising from HK processing propagate bradykinin generation, revealing a novel regulatory level for the kallikrein-kinin system.     

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.     

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.     

高分子量激肽原富含组氨酸区域抑制细胞伸展的机制分析

活化型高分子量激肽原 (activehighmolecularweightkininogen ,HKa)是组织培养板上体外连接蛋白 (vitronectin ,VN)促使细胞伸展的潜在抑制物 ,已证实轻链的富含组氨酸区域 (histidine richdomain ,HRD)是HKa抗细胞伸展的活性区域 .HK的重组HRD (r HRD)能够促使成纤维细胞伸展 .通过基于HRD序列的选择肽分析 ,定位了HRD的细胞伸展序列 .5个肽中的 3个能够使TIG 3细胞伸展 .P 1肽引起的细胞伸展能够被可溶性P 5肽或HKa所抑制 .P 2肽不能抑制P 1或P 5肽引起的细胞伸展 .r HRD以及 3种肽介导的细胞伸展能够被RGD合成肽以及抗αvβ3或α5β1整合素抗体所抑制 .结果提示 ,选择肽引起的细胞伸展是由整合素介导的 ,尽管此区域不含有RGD序列     

活化型高分子量激肽原潜在的抗肿瘤作用及其分子机制

高分子量激肽原是血浆中一种多功能的糖蛋白,与血液凝固的启动、 补体反应及炎症发生等有密切关系.新近的研究显示,活化型高分子量激肽原 具有潜在的抗肿瘤作用.本文综述活化型高分子量激肽原在细胞粘附和血管 生成中发挥的抑制作用及其活性区域,抑制细胞迁移、增殖并诱导细胞凋亡 的作用,及其在细胞表面的作用位点和分子机制.活化型高分子量激肽原作用 机制,包括抑制细胞DNA的从头合成,使细胞周期蛋白D1表达下降,以及通过 影响细胞内信号通路发挥其活性效应等.深入研究活化型高分子量激肽原在 细胞表面作用的信号转导通路可能是今后抗肿瘤研究途径之一.     

Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen

We (8) reported that the cleaved high-molecular-weight kininogen (HKa) and its domain 5 (D5) inhibited angiogenesis. Further studies (15) revealed that D5 could inhibit cell proliferation and induce apoptosis of proliferating endothelial cells, which together may represent a critical part of antiangiogenic activity of HKa and D5. In the present study, we further examined the effect of HKa on cell cycle progression and cell viability. We report that HKa induced a significant upregulation of Cdc2 and cyclin A in proliferating endothelial cells, concurrent with a marked increase of Cdc2 activity. The increased expression of Cdc2 and cyclin A by HKa was not associated with an apparent change in cell cycle profiles of basic fibroblast growth factor-stimulated proliferating cells, but closely correlated with a marked increase of apoptosis, suggesting that the elevated Cdc2 activity is involved in HKa-induced apoptosis of proliferating endothelial cells. Our results support an emerging hypothesis that Cdc2 and cyclin A are important regulators for cell cycle as well as for apoptosis.     

Evidence for binding of the ectodomain of amyloid precursor protein 695 and activated high molecular weight kininogen

To identify ligands that bind to the N-terminal portion of human amyloid precursor protein (APP), we sought binding partners for a fragment of the ectodomain of human APP695 (sAPP(695)T). The probe bound to fragments of high molecular weight kininogen (HK) in rat cortical membrane preparations in vitro. Laser confocal microscopy indicated that APP and HK colocalize near cerebral blood vessels, in the neuropil, and in many neurons of rat brain. sAPP(695)T bound to human activated kininogen (HKa) (K(d)=0.3+/-0.1 nM), but not to inactivated or low molecular weight kininogen. Binding was specific for the light chain sequence of HKa. Biotinylated human HKa also bound to sAPP(695) (K(d)=0.3+/-0.5 nM). sAPP(695) and HKa form tight complexes in solution that can be coimmunoprecipitated. These results support the hypothesis that forms of APP and kininogen can interact in brain tissue. Considering the implications of APP in neurite outgrowth, the APP-HKa interaction could modulate neurogenesis.     

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.     

Assembly and activation of HK-PK complex on endothelial cells results in bradykinin liberation and NO formation

Prekallikrein (PK) activation on human umbilical endothelial cells (HUVEC) presumably leads to bradykinin liberation. On HUVEC, PK activation requires the presence of cell-bound high-molecular-weight kininogen (HK) and Zn(2+). We examined the Zn(2+) requirement for HK binding to and the consequences of PK activation on endothelial cells. Optimal HK binding (14 pmol/10(6) HUVEC) is seen with no added Zn(2+) in HEPES-Tyrode buffer containing gelatin versus 16--32 microM added Zn(2+) in the same buffer containing bovine serum albumin. The affinity and number of HK binding sites on HUVEC are a dissociation constant of 9.6 +/- 1.8 nM and a maximal binding of 1.08 +/- 0.26 x 10(7) sites/cell (means +/- SD). PK is activated to kallikrein by an antipain-sensitive mechanism in the presence of HK and Zn(2+) on HUVEC, human microvascular endothelial cells, umbilical artery smooth muscle cells, and bovine pulmonary artery endothelial cells. Simultaneous with kallikrein formation, bradykinin (5.0 or 10.3 pmol/10(6) HUVEC in the absence or presence of lisinopril, respectively) is liberated from cell-bound HK. Liberated bradykinin stimulates the endothelial cell bradykinin B2 receptor to form nitric oxide. Assembly and activation of PK on endothelial cells modulates their physiological activities.