Corneal activation of prothrombin to form thrombin, independent of vascular injury.
OBJECTIVE
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.
Ayala
Aidee
A
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Warejcka
Debra J
DJ
Olague-Marchan
Monica
M
Twining
Sally S
SS
D001779:Blood Coagulation Factors / Q000502:physiology*; D015153:Blotting, Western; D002166:Camptothecin / Q000494:pharmacology; D002199:Capillary Permeability; D002478:Cells, Cultured; D003315:Cornea / Q000502:physiology*; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004730:Endothelium, Vascular / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005344:Fibrinopeptide A / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D016850:Limbus Corneae / Q000098:blood supply; D009599:Nitroprusside / Q000494:pharmacology; D010314:Partial Thromboplastin Time; D011516:Prothrombin / Q000235:genetics / Q000378:metabolism*; D011517:Prothrombin Time; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013917:Thrombin / Q000096:biosynthesis*
D001779:Blood Coagulation Factors; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D009599:Nitroprusside; D005344:Fibrinopeptide A; D011516:Prothrombin; D013917:Thrombin; D002166:Camptothecin