α2-Macroglobulin-Kallikrein Complexes Detect Contact System Activation in Hereditary Angioedema and Human Sepsis

Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the α2- macroglobulin-kallikrein (α2M-Kal) complex using an F(ab’)2 derivative of a monospecific polyclonal antibody against α2M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4°C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for α2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of α2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for α2M-Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks

Kininogen Binding via Specific Urokinase Receptor Domains Binding of High Molecular Weight Kininogen to Human Endothelial Cells Is Mediated via a Site within Domains 2 and 3 of the Urokinase Receptor

Abstract The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125 I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the ␤ chain of the endothelial cell vitronectin receptor (integrin ␣ v ␤ 3 ), or fibrinogen, another ␣ v ␤ 3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.