Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm
Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer
Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer
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Research Article Neuroscience Vascular biology

Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm

Abstract

Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH), defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity, and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO signaling. We identified 2 pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.

Authors

Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer

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Figure 1

Haptoglobin restores Hb-disrupted nitric oxide signaling in cerebral arteries.

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Haptoglobin restores Hb-disrupted nitric oxide signaling in cerebral art...
(A) K-means clustering of CSF proteins of 3 patients collected at days 1, 4, 7, 11, and 14 after aSAH. The right panel shows a principal component analysis of the identified proteins. Cluster 1: proteins remaining unchanged. Cluster 2: proteins decreasing over time. Cluster 3: proteins increasing over time. ALB, albumin; HP, haptoglobin; HPR, haptoglobin related protein; HBB, Hb-beta; HBA, Hb-alpha; FTH, ferritin heavy-chain; HBD, Hb-delta; CAT, catalase; CA1, carbonic anhydrase; FTL, ferritin light-chain. (B) Image and absorption spectrum of a (centrifuged) patient-derived CSF sample before (red) and after (green) selective removal of Hb. Erythrolysed CSF disrupts the dilatory NO response of porcine basilar arteries to MAHMA-NONOate. NO signaling is restored after removal of Hb from the CSF (n = 12, group mean ± SD). (C) Linear regression of CSF-protein ion intensities before and after passing through the haptoglobin-affinity column. (D) Tension traces of porcine basilar artery segments immersed in preerythrolysed (n = 1 patient; blue) and in erythrolysed (n = 2 patients; red/magenta) CSF from aSAH patients. Baseline and after sequential addition of MAHMA-NONOate, haptoglobin, MAHMA-NONOate, and L-NIO. Thick lines represent the mean recordings of replicate artery segments. (E) Relative changes of the steady-state tension of arteries immersed in patient-derived erythrolysed CSF after addition of haptoglobin (equimolar to cell-free Hb). The diamonds represent the mean and 95% CI (n = 8 patient samples). (F) NO-mediated dilation of arteries immersed in CSF from aSAH patients (n = 8) after administration of MAHMA-NONOate. Arteries were sequentially probed in preerythrolysed CSF (blue), erythrolysed CSF (red), and after the addition of haptoglobin to the erythrolysed CSF (green). (G) Size exclusion chromatography of CSF analyzed in E and F before (Hb) and after addition of haptoglobin (Hb-Hp).