Germinal Matrix Hemorrhage (GMH) is a devastating neurologic pathology in neonates that leads to impaired neurodevelopmental processes and approximately 90% rate of morbidity and mortality within two years in severe cases. Recent studies in our lab have indicated a role for complement in the initiation and propagation of secondary injury after GMH, which includes neuroinflammation and hydrocephalus development. Here we investigate a strategy to target complement inhibition specifically to sites of P-selectin expression, a relevant adhesion molecule at sites of vascular injury/inflammation, as well as investigate the role of P-selectin in leukocyte recruitment and the propagation GMH secondary injury. We prepared two fusion proteins consisting of anti-P-selectin single chain antibodies (scFv) linked to Crry, a complement inhibitor. One of the scFv targeting vehicles (2.12scFv) additionally blocked the cell adhesion site of P-selectin, whereas the other (2.3scFv) bound P-selectin without blocking its function. For our investigations, post-natal mice on day 4 (P4) were subjected to collagenase induced-GMH and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. Histopathological and behavioral analyses were performed at P14 and P45. After GMH injury, 2.3Psel-Crry treatment resulted in reduced infarct size and neurological deficits with improved survival, whereas 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. MRI analyses revealed that 2.3Psel-Crry also reduced post-hemorrhagic hydrocephalus (PHH) development. We also found that 2.12Psel-Crry, but not 2.3Psel-Crry, had an anti-coagulative effect as determined by increased bleeding time and decreased heterotypic platelet interactions. P-selectin expressed on platelets is known to have a pro-coagulative function, and this effect of 2.12Psel-Crry likely explains the worse outcomes when used to treat this hemorrhagic condition. In summary, we show that GMH induces expression of P-selectin, the targeting of which with a complement inhibitor is protective against secondary neuroinflammation and development of PHH. Although unexpected, the worsened outcomes with a targeted construct that also inhibits P-selectin function (ie. 2.12Psel-Crry) can be explained by its effect on interfering with the coagulation cascade. Whereas the 2.3Psel-Crry construct has potential for protecting against the pathogenic sequelae of GMH, the 2.12Psel-Crry construct has promising potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.
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