ALBERT

Description: Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent vβ3 integrin–mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with vβ3 integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn2+-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN (“somatomedin B region”). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn2+-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys–rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions.

Description: The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

Description: We have demonstrated that high molecular weight kininogen (HK) binds specifically on endothelial cells to domain 2/3 of the urokinase receptor (uPAR). Inhibition by vitronectin suggests that kallikrein-cleaved HK (HKa) is antiadhesive. Plasma kallikrein bound to HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell binding domains would inhibit angiogenesis. We found that recombinant domain 5 (D5) inhibited endothelial cell migration toward vitronectin 85% at 0.27 μM with an IC50 (concentration to yield 50% inhibition) = 0.12 μM. A D5 peptide, G486-K502, showed an IC50 = 0.2 μM, but a 25-mer peptide from a D3 cell binding domain only inhibited migration 10% at 139 μM (IC50 〉 50 μM). D6 exhibited weaker inhibitory activity (IC50 = 0.50 μM). D5 also potently inhibited endothelial cell proliferation with an IC50 = 30 nM, while D3 and D6 were inactive. Using deletion mutants of D5, we localized the smallest region for full activity to H441-D474. To further map the active region, we created a molecular homology model of D5 and designed a series of peptides displaying surface loops. Peptide 440-455 was the most potent (IC50 = 100 nM) in inhibiting proliferation but did not inhibit migration. D5 inhibited angiogenesis stimulated by fibroblast growth factor FGF2 (97%) in a chicken chorioallantoic membrane assay at 270 nM, and peptide 400-455 was also inhibitory (79%). HK D5 (for which we suggest the designation, “kininostatin”) is a potent inhibitor of endothelial cell migration and proliferation in vitro and of angiogenesis in vivo.

Description: Objectives: This study examines the role of pericardial wound monocytes in thrombin generation during clinical cardiac surgery with cardiopulmonary bypass (CPB). Background: The mechanism by which wound mononuclear cells rapidly express procoagulant activity is unexplained. Methods: Factor VII activation (FVIIa) was measured using recombinant, truncated, soluble tissue factor (rsTF) and various blood cells in vitro. FVIIa was also measured with monocytes and soluble plasma tissue factor taken before CPB and simultaneously from the pericardial wound and perfusion circuit during CPB in thirteen patients. Results: RsTF in combination with monocytes, but not platelets, neutrophils or red cells, accelerates activation of FVII beginning at 1 pmole/L rsTF. Less than 1% rsTF is bound, yet catalytic activity peaks at 7 minutes and decays afterwards. In wound plasma, monocytes are activated (MCP-1 = 29.5 ± 2.1 pmoles/L) and wound plasma tissue factor (wpTF) is substantially elevated (3.64 ± 0.45 pmoles/L) with 81.7% in the supernatant and 18.3% in microparticles. By Western blot all forms of plasma TF migrate at Mr 65 kDa [TF/FVII(FVIIa) complex]. Wound monocytes and C5a activated prebypass or perfusate monocytes plus wpTF convert all available FVII to FVIIa. Activated monocytes plus supernatant TF/FVII(VIIa) more efficiently activate factor X than microparticle TF/FVII(FVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wpTF is 0.944 (p = 0.0004). Conclusions: During clinical cardiac surgery with CPB wound monocytes plus wpTF or microparticle-free, protein fragments of wound tissue factor preferentially accelerate activation of FVII and FX. This system represents a new mechanism of thrombin generation.

Description: 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 Kd = 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 (kon and koff, respectively) were determined, and they yielded a dissociation constant (Kd) of 3.2×10−9mol/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.

Description: We have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer. (Blood. 2004;104:2065-2072)

Description: The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

Description: Phosphorylation /activation of Akt is a critical event in platelet activation stimulated by thrombin. We previous demonstrated that the activation of PDE3A was increased concomitantly with the phosphorylation /activation of Akt. In order to demonstrate a link between these two events, in this study, we monitored thrombin-induced cAMP changes in washed platelets. We confirmed that the platelet cAMP level decreased following thrombin stimulation. The thrombin-induced decrease of cAMP was inhibited by an Akt specific inhibitor (1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate). This Akt inhibitor exhibited a concentration-dependent inhibition of thrombin-induced decrease of cAMP. The Akt inhibitor blocked 70 % of thrombin-induced decrease of cAMP. Moreover, a phosphoinositide 3-kinase inhibitor wortmannin also reduced the thrombin effect, consistent with its upstream position relative to Akt. These results suggested that phosphorylation /activation of Akt is required for decrease of cAMP in platelets. In addition, we found that thrombin-induced cAMP level changes were markedly reduced following preincubation of platelets with milrinone, a selective PDE3A inhibitor. We therefore examined PDE3A activity after stimulation of platelets with thrombin with or without the Akt inhibitor or wortmannin. Either the Akt inhibitor or wortmannin inhibited the thrombin-induced activation of PDE3A. Our data indicated that phosphorylation/activation of Akt plays a major role in regulation of cAMP by thrombin. The activation of Akt by thrombin results in activation of PDE3A and a consequent decrease of the intracellular cAMP level, which serves as a positive feedback enhancing the thrombin activation of platelet function. Understanding the mechanisms that are involved in regulation of PDE3A in platelets could provide new targets for therapeutic advances in the treatment of thrombotic disorders.

Description: The HLA-B27 rat is a well-characterized model of human bowel disease, rheumatoid arthritis and psoriasis. Previous studies of chronic inflammation in other rat models of inflammatory disease have demonstrated activation of the kallilrein-kinin system (KKS) as well as modulation by a kallikrein inhibitor and HK deficiency. The effects of C11C1, a monoclonal antibody acting to inhibit cellular binding of high molecular weight kininogen (HK), were studied in the HLA-B27 transgenic rat. Thrice weekly intraperitoneal injections of C11C1 (1.9 mg/kg) or IgG1 (6mg/kg) were given to male HLA-B27 transgenic rats for 3 weeks, beginning when the rats were 23 weeks old. Stool character was scored daily as a measure of intestinal inflammation, and the rear limbs were scored daily for clinical signs of arthritis, tarsal joint swelling and erythema. At the end of the experiment the animals were euthanized, and the colon and tarsal joint histologic lesions were examined. The histology sections were assigned a numerical score for colonic inflammation, synovitis, and cartilage damage. Activation of KKS was assessed by assays of plasma prekallikrein, HK, factor XI, and factor XII. Administration of the monoclonal antibody directed against HK rapidly decreased the clinical scores of inflammatory bowel disease from 3.0 ± 0 to 1.2 ± 0.2 (p

Description: High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained