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. 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. Recent investigations indicate that high molecular mass kininogen (HK) 1The abbreviations used are: HK, high molecular mass kininogen; uPAR, urokinase plasminogen activator receptor; PK, prekallikrein; suPAR, soluble urokinase plasminogen activator receptor; scuPA, single chain urokinase plasminogen activator; HUVEC, human umbilical vein endothelial cell(s). 1The abbreviations used are: HK, high molecular mass kininogen; uPAR, urokinase plasminogen activator receptor; PK, prekallikrein; suPAR, soluble urokinase plasminogen activator receptor; scuPA, single chain urokinase plasminogen activator; HUVEC, human umbilical vein endothelial cell(s). binds to endothelial cell membranes through an interaction with a multiprotein receptor complex comprising at least cytokeratin 1, gC1qR, and the urokinase plasminogen activator receptor (uPAR) (1Hasan A.A.K. Zisman T. Schmaier A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3615-3620Crossref PubMed Scopus (162) Google Scholar, 2Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 4Joseph K. Grebrehiwet B. Peerschke E.I.B. Reid K.B.M. Kaplan A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8552-8557Crossref PubMed Scopus (214) Google Scholar, 5Colman R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). The three proteins co-localize on the endothelial cell membrane (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). The same three proteins form a receptor complex for factor XII (7Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2002; 99: 3585-3596Crossref PubMed Scopus (143) Google Scholar), but binding of factor XII in vivo is likely limited both by the low plasma concentration of free Zn2+, which is below the requirement for FXII binding and by the much higher plasma concentration of HK (7Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2002; 99: 3585-3596Crossref PubMed Scopus (143) Google Scholar). Binding of HK to this multiprotein receptor complex predominates, localizing prekallikrein (PK) to the cell surface. The plasma concentration of PK and the ambient free Zn2+ concentration in plasma also prevent FXI from binding to HK under conditions where platelets or other cells are not activated (8Mahdi F. Shariat-Madar Z. Schmaier A.H. J. Biol. Chem. 2003; 278: 43983-43990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). PK bound to HK on endothelial cells is proteolyzed by membrane-expressed prolylcarboxypeptidase to form plasma kallikrein (9Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 2002; 277: 17962-17969Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 10Moreira C.R. Schmaier A.H. Mahdi F. da Motta G. Nader H.B. Shariat-Madar Z. FEBS Lett. 2002; 523: 167-170Crossref PubMed Scopus (54) Google Scholar). This multiprotein receptor complex thereby regulates the assembly and activation of the plasma kallikrein/kinin system. The requirements for HK binding to each component of this receptor complex and the effects of other biologically relevant ligands, e.g. urokinase, on this binding have not been well delineated. It is known that both the heavy and light chains of HK interact with a region of cytokeratin 1 coded by exon 1 (2Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Antibody to this region completely inhibits HK binding to cytokeratin 1 as well as to the receptor complex (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). Likewise, some antibodies to gC1qR and uPAR completely block HK binding to the proteins individually as well as when they are part of the complex expressed on endothelial cells (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar, 8Mahdi F. Shariat-Madar Z. Schmaier A.H. J. Biol. Chem. 2003; 278: 43983-43990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, it is unclear whether each component of the cellular complex recognizes discrete or overlapping portions of HK and whether each molecule of HK binds to more than one component of the complex at the same time. To begin to address these issues, we sought to identify the regions in HK and uPAR required for binding. The results indicate the existence of multiple potential sites of interaction between this ligand and receptor and provide insight into the assembly of HK on its multireceptor complex and the influence of urokinase on this interaction. Proteins and Materials—Single-chain and two-chain HK (specific activity of 13-17 units/mg and 17 units/ml, respectively) in 4 mm sodium acetate-HCl and 0.15 m NaCl, pH 5.3, plasma kallikrein, and PK (specific activity of 22-27 units/mg) in 4 mm sodium acetate-HCl and 0.15 m NaCl, pH 5.3, were purchased from Enzyme Research Laboratories, Inc. (South Bend, IN). Carboxymethylated papain was prepared as previously reported (11Meloni F.J. Schmaier A.H. J. Biol. Chem. 1991; 266: 6786-6794Abstract Full Text PDF PubMed Google Scholar). 56-kDa (56HKa) and 46-kDa (46HKa) kallikrein-cleaved HK was prepared by dialyzing 1 mg of single-chain HK into HEPES carbonate buffer (137 mm NaCl, 3 mm KCl, 12 mm NaHCO3, 14.7 mm HEPES, 5.5 mm dextrose containing 2 mm CaCl2, and 1 mm pH plasma kallikrein in a of to HK was F.J. Schmaier A.H. Blood. PubMed Google Scholar). The was at for of and the was by from each were by the of to and 46-kDa HK was were at the and the were by 1 mm The HK was HEPES carbonate pH to the The of the HK was on a and the concentration was the A and were by low molecular mass and were purchased from were with uPAR was by F. Peptides and peptides to domains of HK were at the and of the of as previously reported H. A.A.K. J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar, A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.A.K. H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and overlapping peptides to each domain of suPAR were at of the peptides from suPAR are upon the the acid of The peptides used were and as by high and mass A uPAR was by F. from the of M. A. Clin. PubMed Scopus Google Scholar). antibodies and were the of A. Mazar peptides and influence to binding to is for the three of the the acid the of in the the acid the of the HK its the IC50 of biotin-HK or binding to suPAR by each of the is for the three of the the acid the of in the the acid The the of the HK its A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.A.K. H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google The the IC50 of biotin-HK or binding to suPAR by each of the in a and overlapping peptides from uPAR and influence on biotin-HK binding to suPAR or endothelial is for the three of the the acid the of in the the acid the of the of urokinase plasminogen activator receptor the the IC50 of biotin-HK binding to suPAR or endothelial cells by each of the peptides prepared from the of is for the three of the the acid the of in the the acid The the of the of urokinase plasminogen activator receptor the The the IC50 of biotin-HK binding to suPAR or endothelial cells by each of the peptides prepared from the of in a of and urokinase plasminogen activator receptor domains of suPAR (1Hasan A.A.K. Zisman T. Schmaier A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3615-3620Crossref PubMed Scopus (162) Google Scholar, 2Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), a containing soluble suPAR domains 1 and 2 and 3 and were prepared and expressed the to as previously K. A. Mazar A. W. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. A.H. Mazar A. Blood. 2003; PubMed Scopus Google Scholar). domains and 3 of suPAR were prepared from by sequential with and as previously reported Mazar A. Wang J. R. R. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of suPAR in was with the K. A. Mazar A. W. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The suPAR domain 2 and 3 are in and were from the a or affinity was from the a and was by and K. A. Mazar A. W. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). suPAR are upon the of suPAR the is for the three of the the acid the of in the the acid The the acid of the uPAR that the of the is for the three of the the acid the of in the the acid The the acid of the uPAR that the of the in a and proteins were and to membranes at for 1 The were in buffer 0.15 m NaCl, and mm pH for 1 A.H. A. R. J. Clin. Google Scholar). The membranes were with to uPAR at for 1 The membranes were and with for 1 and binding was with an from of the were at umbilical vein endothelial cells endothelial cell and were purchased from The cells were to the The cells between the and were to the of the A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was The cell were by on a and mg of HK in was m sodium 0.15 m NaCl, pH A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of was for 2 on the was HK was by at an of for HK and a a activity of 46HKa was in the same Peptides and were with at suPAR and HK were with at a of of of to K. A. Mazar A. W. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by or Binding proteins or peptides in the or of proteins or peptides in HEPES carbonate pH (8Mahdi F. Shariat-Madar Z. Schmaier A.H. J. Biol. Chem. 2003; 278: 43983-43990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), were to with of or with other proteins or peptides by an at 4 in m sodium pH the with the were with HK, or peptides for 1 at The were three with HEPES carbonate pH The bound proteins or peptides were and by the of the at a as previously A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.A.K. Schmaier A.H. Blood. PubMed Google Scholar). Binding of HK to was in m sodium 0.15 m NaCl, pH at 4 at a concentration of 1 Binding of to suPAR was in the of of HK in HEPES carbonate binding to cells was as previously reported K. A. Mazar A. W. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on of peptides from suPAR to block PK activation on endothelial cells was were as in HK and PK were to the in the or of or from domains of suPAR in HEPES carbonate pH for 1 at three with HEPES carbonate pH mm was and of the was the 1 at G. R. A.A.K. Schmaier A.H. Blood. Google Scholar). of HK of HK, and was under and HK, 56HKa, and 46HKa predominantly at at and and at and results indicate that and 46HKa heavy chains and light HK, and 46HKa as single at 110, and with of a in the molecular mass of plasma HK when it is activated by plasma kallikrein A.H. A. R. J. Clin. Google Scholar). Binding of to and whether these of HK in affinity for or for suPAR. investigations were with HUVEC. HK, 56HKa, and 46HKa blocked the binding of HK to with IC50 of and nm, HK, 56HKa, and 46HKa blocked biotin-HK binding to suPAR with IC50 of 60, 110, and 8 nm, indicate that 46HKa is a more of the binding of HK to and suPAR than are the activated of This was with previously reported that kallikrein-cleaved HK bound more to uPAR than HK R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). The also that HK also bound to suPAR, with lower affinity than its This that of HK its binding to suPAR. This in the affinity of binding for 46HKa was on HUVEC, which binding sites for the Binding for suPAR within of been in that HK bound to cells through within both its light and heavy chains H. A.A.K. J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar, A.A.K. H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we sought to identify the in domain of HK that bound to suPAR and inhibited the binding of HK and 46HKa H. A.A.K. J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar, A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.A.K. H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Binding of biotin-HK to suPAR was blocked by peptides and with IC50 of and was a of binding Peptides and inhibited binding to suPAR with IC50 of and indicate that the region to of domain of HK the affinity for suPAR, other portions of domain also the to Binding for suPAR within 3 of was with peptides within the region in domain 3 of HK H. A.A.K. J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Google Peptides and inhibited biotin-HK binding to suPAR with IC50 of and from the amino-terminal end of this domain was The a domain inhibited the binding of biotin-HK to suPAR with an IC50 of not and inhibited binding to suPAR with IC50 of and was an of and biotin-HK binding not these that HK bound to suPAR through regions on both its heavy and light the binding of suPAR to domain peptides was at least more the IC50 of with This was by the that and bound to suPAR Binding for HK in the binding sites in suPAR for a the by a that blocked HK binding to endothelial cells (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar, M. A. Clin. PubMed Scopus Google was bound to suPAR domain 2 and to fragments containing domains 2 3 and bound to but not to under the same conditions are with in which it was reported that a to domains 2 and 3 of uPAR blocked HK binding to R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar, F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). investigations were to whether HK bound to domains 2 and 3 of suPAR R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). S-D2D3 inhibited the binding of biotin-HK and -46HKa to suPAR by the same domain 1 of suPAR blocked binding S-D1 and S-D2D3 blocked biotin-HK and -46HKa binding to an than that with suPAR. on these we on the regions with domains 2 and 3 of of biotin-HK and -46HKa binding to suPAR. were with 1 of suPAR. biotin-HK or in HEPES carbonate buffer was for 1 in the or of 1 HK or suPAR, soluble or soluble domains 2 and 3 or a of soluble domains S-D1 and the were and the of biotin-HK or 46HKa bound was The the of three or more Binding for HK within 2 and 3 of the within this of suPAR that HK, a of acid peptides were at the of domain 1 and domains 2 and 3 in the of domain inhibited binding of biotin-HK with an IC50 of overlapping peptides that or the region, and inhibited biotin-HK binding to suPAR with IC50 of and 1 that suPAR a binding site for HK within of domain This is biologically these peptides also inhibited biotin-HK binding to Binding of biotin-HK to also was inhibited by and with IC50 of and 4 A was to begin to within suPAR domain 3 peptides from the amino-terminal portion of domain 3 of uPAR, and inhibited biotin-HK binding to with IC50 of 12 and that an site also was within of domain 3 of Binding of a uPAR 2 3 to on its to HK binding to suPAR, we whether from suPAR bound to bound to HK to in a and Binding of to HK was blocked by HK and or also bound to HK and or blocked the binding of to HK that regions on domains 2 and 3 to form an the in suPAR Using a to identify the binding sites for HK in domains 2 and 3 of suPAR, was on the in regions that been in binding on The suPAR its to biotin-HK binding to suPAR and suPAR inhibited biotin-HK binding with an IC50 of a to suPAR and results that to HK binding to suPAR. To this was to the more than to suPAR at inhibited biotin-HK binding suPAR but than the and that was for the interaction. were also with the in and that the carboxyl-terminal region of domain 2 from to the region on suPAR. with this suPAR and that been previously to single chain urokinase with affinity and nm, for K. A. Mazar A. W. S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google inhibited the binding of biotin-HK to suPAR with IC50 of 1 and with the with suPAR investigations that urokinase not block HK binding to suPAR R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar), more the region as a urokinase-binding site J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). with these inhibited the binding of biotin-HK to suPAR with an IC50 of were with binding to suPAR not suPAR and were of binding to cells not also bound urokinase with a affinity not results that the site in domain 2 of suPAR in the binding of domain of HK overlapped with the binding site for urokinase. The affinity of for suPAR is than that of were also to the binding site for HK in domain 3 of suPAR. The suPAR not biotin-HK binding to suPAR and in region inhibited biotin-HK binding with an IC50 of to that of suPAR. to the of acid in domain 3 for binding of HK, an acid with the results of the in of suPAR Peptides on whether peptides from suPAR that bound HK with PK activation on endothelial cells A.A.K. Schmaier A.H. Blood. PubMed Google Scholar). inhibited PK activation on by with its to HK binding to these cells Likewise, peptides and that blocked HK binding to suPAR with IC50 of and also inhibited PK activation on HK by and of HK binding by peptides to the region on suPAR blocked PK activation on the of the suPAR and an for uPAR in PK investigations address the by which the HK and PK complex on the multiprotein HK receptor complex on endothelial indicate that the binding of HK to uPAR is through regions on both its domain 3 heavy chain and domain light results are with which that domains 3 and of HK are in cell binding H. A.A.K. J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar, A.A.K. J. Schmaier A.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It is of that the domain region of HK, which its and binds to suPAR with a higher affinity than the domain 3 region, that uPAR to these biological It been reported previously that HK not to suPAR in to HK R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). results are at with that biotin-HK bound to uPAR on endothelial cells (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). This by in the that HK or binds suPAR with lower affinity than of HK the and an amino-terminal portion from the light chain of HK its binding to uPAR than the of a receptor F. Mazar A.P. McCrae K.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). have for the and activity of HK, which under is in to is likely to binding to uPAR under conditions F. Mazar A.P. McCrae K.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, K. R. Z. J. Wang McCrae K.R. J. PubMed Scopus Google Scholar, R.W. Pixley R.A. J. 2003; PubMed Scopus Google Scholar). from HK activity R.W. Pixley R.A. J. 2003; PubMed Scopus Google Scholar, The Google Scholar). it is likely that inhibits in where of these fragments have been to the influence of plasma the of HK to its and F. Mazar A.P. McCrae K.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, K. R. Z. J. Wang McCrae K.R. J. PubMed Scopus Google Scholar, R.W. Pixley R.A. J. 2003; PubMed Scopus Google Scholar, The Google Scholar). The region within domain 2 of uPAR with a region in the binding of J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google to a R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar), we that inhibited the binding of HK to suPAR. We this to the of we that several of or as The plasma concentration of HK is and that of nm, that HK the binding of to uPAR in the other we and have that plasma kallikrein is a activator of and that endothelial cell membranes provide a for this G. R. A.A.K. Schmaier A.H. Blood. Google Scholar, A. K. T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This by the that a affinity for uPAR than at plasma of the to by scuPA, and the to by of uPAR by when cells have been to and of A. R. F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google on endothelial cells to or binding of HK to the other of this multiprotein receptor complex activation of bound to uPAR G. R. A.A.K. Schmaier A.H. Blood. Google Scholar). Recent investigations indicate that cytokeratin 1 with uPAR gC1qR but that uPAR not to gC1qR on endothelial cells (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar, K. B. Kaplan A.P. PubMed Google Scholar). 1 with uPAR, of PK on HK for activation by prolylcarboxypeptidase to to uPAR where it a plasma kallikrein The investigations also indicate that each domain of suPAR the to at least one portion of HK and that binding. are to whether the of domain 1 is to high affinity binding sites in domain 2 and 3 or whether each domain in uPAR recognizes a on HK a binding that HK to each domain of uPAR that HK or interacts with uPAR than it with cytokeratin the 1 interaction where HK binds to one portion of cytokeratin 1 by exon 1, HK and uPAR each multiple potential sites of sites. it is not known whether HK interact with of these sites to or a portion of HK to uPAR, other HK regions to to the same or other or other of the multiprotein these investigations provide insight into the of the multireceptor complex for of with one or more of this complex likely to its biological the of HK to as an binding by its domain 3 to and domain to regulates T. S. Pixley R.A. R.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). with its other binding proteins for activation of the and and of uPAR as a between and HK binding to plasma and the of