The initial hypothesis that uPAR may mediate the apoptotic effect of HKa derived from our study showed that HKa binds to endothelial cells through D2 and D3 of uPAR . The binding of HKa to endothelial cells can be completely blocked by an anti-uPAR D2 and D3 antibody or by soluble recombinant uPAR. Therefore, uPAR could be a logical target of HKa action. The uPAR–gC1qR bimolecular complex may provide a cell surface structure for the assembly of HK/prekallikrein and subsequent generation of HKa and BK [11,12]. This complex may also mediate the effect of HKa in inhibiting cell proliferation and induction of apoptosis. However, this concept was challenged by one study  because none of three antibodies against gC1qR, CK1 or uPAR, which have been previously shown to inhibit HK or HKa binding to endothelial cells [7,8,10], affected the ability of HKa to induce apoptosis of endothelial cells when fibronectin was used to coat the plates. However, in a recent investigation , we were able to show that HKa or D5 completely inhibited uPAR-mediated cell adhesion to vitronectin but not to fibronectin in U937- and uPAR-transfected BAF-3 cells and thereby promoted cell detachment. The antiadhesive effects of HKa and D5 in these cell systems are also critically dependent on the ECM proteins on which the cells were plated . Taken together, these studies suggested that uPAR could be the target of HKa but may require the presence of vitronectin. Unique functions of uPAR specifically associated with vitronectin have been well documented in other types of cells. For example, uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA but require uPAR binding to vitronectin . In fibrosarcoma (HT-1080) cells, uPA activity is significantly higher in the cells grown on vitronectin than in those grown on fibronectin. Under these conditions, uPAR were detected as clusters in the focal adhesion contacts in the cells grown on vitronectin, but were evenly distributed in the cells grown on fibronectin , indicating that the expression as well as the distribution of uPAR, thus its function, is affected by the nature of ECM proteins. Similar patterns of uPAR distribution as found in fibrosarcoma were observed in endothelial cells (unpublished data). These results may explain why HKa inhibited cell adhesion to vitronectin but not to fibronectin. Vitronectin–uPAR interactions, which are concentrated in the focal contact areas (the most critical part for cell adhesion), play an important role in cell adhesion to vitronectin. On the other hand, uPAR may play a less important role in cell adhesion to fibronectin because of their uniform distribution throughout cells grown on fibronectin, especially in the absence of its ligand, vitronectin. In addition, fibronectin is the ligand for at least 12 integrins, while vitronectin and gelatin (denatured collagens) are the ligands for only four (αvβ, αvβ3, αvβ5, αIIbβ3) and three integrins (αvβ3, α5β1, αIIbβ3), respectively . The larger number of integrins binding to fibronectin probably contributes to the resistance of some cells to the antiadhesive effect of HKa.
Our most recent study  provided more convincing evidence to support the role of uPAR in mediating the effect of HKa. We showed that the apoptotic effect of HKa was blocked by three different anti-uPAR antibodies in cells grown on vitronectin. Further results revealed that uPAR formed a signaling complex containing integrins αvβ3 or α5β1, caveolin, and Src kinase Yes in endothelial cells. HKa physically disrupted the formation of this complex in a manner that paralleled its apoptotic effect. Together with a previous report , we can now establish a signaling cascade in endothelial cells, vitronectin–uPAR–αvβ3–caveolin–Src–FAK–paxillin, which mediates adhesion, proliferation, and survival of many types of cells. We proposed an action model of HKa (Fig. 2). HKa disrupts this signaling cascade by at least two mechanisms. First, HKa directly binds to the D2 and D3 domains of uPAR through its D5 region, thus preventing the binding of vitronectin to uPAR and its subsequent interaction with integrins as well as other signaling molecules. Second, through its direct binding to the amino terminal region of vitronectin , proximal to the RGD region (integrin binding site), HKa can potentially disrupt the binding of vitronectin to integrins that utilize vitronectin as ligand, such as αvβ3. As a result of either of these mechanisms, cells will not be able to adhere to vitronectin properly and will eventually undergo apoptosis. It should be pointed out that this model is a simplified version of the complex interactions among signaling molecules. Many other molecules, such as α5β1, gC1qR, and CK1, are probably involved but not included in the model for simplicity. We also emphasize that this model does not exclude the possibility that other targets and mechanisms of HKa action may exist in endothelial cells in addition to uPAR and vitronectin. For example, HKa can directly bind to Mac1 integrin found in leukocytes, thus blocking adhesion of HEK293 cells transfected with Mac1 to fibrinogen and intercellular adhesion molecule-1 . Therefore, HKa may potentially disrupt integrin-mediated adhesion in a similar manner through its direct association with certain integrins in endothelial cells. It was recently reported that the apoptotic activity of HKa may be mediated through its interaction with tropomyosin . In addition, HK has been shown to bind heparin sulfate proteoglycans at the endothelial cell surface , which may also be involved in mediating the effect of HK/HKa. However, other studies demonstrate that complete removal of sulfated mucopolysaccharides by heparinases did not alter HK binding . Apparently, the interaction between HKa and cell surface proteins is highly complex and requires further investigation.