In previous studies, we found that a single injection of PK alone into the vitreous induced both acute RVP and focal areas of sustained RVP at 24 hours postinjection.
13 Since PK is normally bound to HK in plasma, in this study, we injected eyes with the combination of PPK, HK, and FXII to examine the effect of PK in the presence of HK. Comparison with our previous reports
12,13 revealed that PK in the absence or presence of HK exerts similar effects on increasing RVP. In this study, we also observed retinal hemorrhages in eyes receiving two intravitreal injections of PK, whereas hemorrhages were infrequently observed in eyes receiving either saline or bradykinin. Penn et al.
31 reported that multiple dry-needle punctures exert an additive effect to decrease retinal neovascularization in a rodent model of oxygen-induced retinopathy. We have previously reported that a single intravitreal injection of saline in adult rats produced a small but consistent increase in RVP.
4 The appearance of retinal hemorrhage in eyes receiving multiple saline injections, which was not observed in eyes receiving a single saline injection, suggests that consecutive intravitreal injections can exert cumulative effects on BRB dysfunction in adult animals. The absence of an effect of bradykinin on hemorrhage suggests that PK may exert bradykinin-independent effects on the retina. Indeed, recent reports have identified a number of novel PK substrates; for example, PK can function as a plasminogen activator, which could contribute to plasmin-mediated fibrinolysis, activation of matrix metalloproteinases (MMPs), and adipocyte differentiation.
32–34 To search for PK substrates from retinal cells, we exposed culture bovine retinal pericytes to PK for 24 hours and used proteomics to characterize proteins in the conditioned medium. This analysis revealed increased levels of low molecular weight fragments of collagen chain in medium exposed to PK compared with the control. These findings are consistent with a previous study that has shown that PK increased collagen fragments in the secretome from rat brain astrocytes.
22 Since PK has been previously reported to influence plasminogen and MMP activities, the effect of PK on collagen proteolysis could be due to both direct and indirect effects. To examine the potential direct effect of PK on collagen, we incubated purified PK with purified human type IV collagen and used mass spectrometry to analyze individual type IV collagen chains. Although direct comparisons of PK-mediated type IV collagen cleavage fragments obtained from bovine cells and from human placenta is limited, these studies showed that multiple COL4A chains are cleaved by PK and these effects of PK on COL4A cleavage were blocked by a selective PK inhibitor. These findings suggest that PK has a previously unrecognized direct collagenase-like activity. Intracerebral bacterial collagenase injection into the brain is an established experimental model of intracerebral hemorrhage in rodents,
35 and we show that intravitreal injection of collagenase induced retinal hemorrhage. These results suggest that the collagenase-like activity of PK may contribute to its effects on retinal hemorrhage, which requires disruption of the vascular basement membrane.