Faculty Collaboration Database

Corneal activation of prothrombin to form thrombin, independent of vascular injury. Invest Ophthalmol Vis Sci 2007 Jan;48(1):134-43

Date

01/02/2007

Pubmed ID

17197526

DOI

10.1167/iovs.06-0339

Scopus ID

2-s2.0-33846910310 (requires institutional sign-in at Scopus site)   14 Citations

Abstract

PURPOSE: Two major functions of thrombin observed in the cornea are activation of thrombin-sensitive, proteinase-activated receptors and cleavage of fibrinogen to fibrin. The purpose of this study was to determine whether the normal human cornea itself is competent to convert prothrombin to thrombin and synthesizes the mRNA for the proteins required.

METHODS: Human corneas were processed for immunolocalization studies or separated into epithelial, stromal, and endothelial layers for proteins and RNA isolation. The protein extracts were used for Western blots, prothrombin time, and activated partial thromboplastin time assays and fibrinopeptide A generation tests. RNA was used for RT-PCR. Apoptosis of cultured human corneal cells was induced with sodium nitroprusside or camptothecin and activation of prothrombin tested.

RESULTS: Prothrombin and its mRNA were present in all three layers of human donor cornea. It was found to be associated with the cells and the extracellular matrix at similar levels across the cornea. With corneal stromal extracts, activation of either the intrinsic or extrinsic coagulation pathways resulted in thrombin activation and fibrin formation with fibrinopeptide A release. Detection of key components of the coagulation cascades confirmed noninjured human corneas contain factors required for prothrombin activation. In addition, mRNAs for representative factors and inhibitors were detected by RT-PCR and confirmed by sequencing. Apoptotic corneal stromal cells provide a surface for prothrombin activation.

CONCLUSIONS: These studies suggest that the normal avascular human cornea contains and synthesizes the components required for thrombin generation and that this process does not depend on a breech in the limbal vascular endothelium.

Author List

Ayala A, Warejcka DJ, Olague-Marchan M, Twining SS

Author

Sally S. Twining PhD Assistant Dean, Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Blood Coagulation Factors
Blotting, Western
Camptothecin
Capillary Permeability
Cells, Cultured
Cornea
Corneal Stroma
Endothelium, Vascular
Enzyme Inhibitors
Enzyme-Linked Immunosorbent Assay
Epithelium, Corneal
Fibrinopeptide A
Humans
Immunoenzyme Techniques
Limbus Corneae
Nitroprusside
Partial Thromboplastin Time
Prothrombin
Prothrombin Time
RNA, Messenger
Reverse Transcriptase Polymerase Chain Reaction
Thrombin