Factor VIII in the Interstitial Space - Is it there?  How does it get there?  What might it do?


Summary:  There is data to support the presence of factor VIII in the interstitial space arising from basolateral secretion from endothelial cells.  It is possible that factor VIII is involved in the development of interstitial fibrosis.


            I had always considered blood coagulation to be a process which occurred within the vascular space.  I was somewhat surprised to find that the major portion of many plasma proteins including blood coagulation factors was in the extravascular space of which the bulk is interstitial fluid.  While there may be normal function for blood coagulation enzymes such as thrombin in the interstitial space,  there has been more interest in their role in the development of interstitial fibrosis (1,2).  A bit more time in the library resulted in finding several studies where elevated plasma levels of factor VIII were found in patients with fibrosis(3,4).    While most interest focuses on the tissue factor pathway, the observations on elevated factor VIII plasma from patients with fibrosis required some further consideration.  In order for factor VIII to be involved in the etiology of fibrosis, factor VIII needs to be in the interstitial space.
            What is the evidence for factor VIII in the interstitial space?  Factor VIII is a large protein (Mr ≈ 250,000) associated with an even larger protein, von Willebrand factor, in the circulation. As such, factor VIII is most unlikely to pass from the vascular bed to the interstitial space by the process of extravasation.  Factor VIII has been found in lymph (5-8) which is derived from interstitial fluid.  von Willebrand factor (vWF) antigen has been found in peripheral rabbit lymph (7) but was undetectable in peripheral human lymph (8).  The vWF in peripheral rabbit lymph was described as mainly low molecular weight multimers with some high molecular weight material (7).   There is some reason to think that vWF interacts with collagen and other components of the extracellular matrix prevent it from passing into the lymphatic system.   There is no evidence to suggest that large proteins such as factor VIII or vWF, let alone the Factor VIII-vWF complex, can pass from the capillary bed into the interstitial space.  Thus, there must be extravascular secretion of factor VIII and vWF to account their presence in lymph and, by extension, in the interstitial fluid.   Endothelial cells and epithelial cell secrete proteins from the apical surface and from the basolateral surface and both endothelial cells and epithelial cells can demonstrate polarity  such that there may be preferential apical secretion or preferential basolateral secretion. The polarity of cell secretion can be altered by stimuli including inflammation(9). 


            I will start with a discussion of vWF as more is known about the secretion of vWF into interstitial space and while there is no solid evidence, it is likely the that where at least some vWF goes, factor VIII will follow.   Secretion of vWF from endothelial cells can occur at both the apical (luminal) surface and basolateral (subluminal, subendothelial) surface.   van Buul-Wortelboer and coworkers (10)‑ observed polarity in the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells cultured on a collagen lattice.   Constitutive secretion of vWF occurs into both basolateral and apical (luminal) compartments with preferential secretion into the basolateral compartment.  Stimulated (regulated) secretion of vWF stored in Weible-Palade bodies preferentially occurred at the apical or luminal surface.    Differing results were obtained by Sporn and coworkers (11)  who also used human umbilical vein endothelial cells cultured on a polycarbonate membrane.   These investigators observed equal amounts of constitutive basolateral secretion and apical secretion of vWF and stimulation(calcium ionophore, phorbol ester) provided preferential basolateral secretion.   It is possible that the use of collagen as a matrix influenced the polarity endothelial cells secretion as has been previously observed (12).   Regardless, the results suggest that there would be substantial secretion of vWF into the interstitial space.    Rand and coworkers (13) have  reviewed the function of vWF in the interstitial space  with emphasis on the binding  to collagen VI in the extracellular matrix and the role in hemostasis.  The binding of vWF to collagen VI was the subject of earlier work by Denis and coworkers (14).


            While it is tempting to suggest that constitutive synthesis is responsible for the maintenance of normal levels of vWF, this is likely not correct considering the constitutive vWF is low-molecular weight polymers (15) which are not effective in platelet aggregation.   The process of vWF synthesis in the endothelial cells involves the synthesis of a dimer form containing a propeptide domain.  The dimer form is taken into Weible-Palade bodies where large oligomeric forms of vWF are formed released in response to stimuli (16).  The nature  and extent of of constitutive secretion is not clear as there are differences in opinion.  Sporn and coworkers (17) suggest that 90% of the vWF is constitutively secreted which Giblin and coworkers (18) suggest that only a small amount of vWF is released in a constitutive pathway; however, Giblin and coworkers do  state that the majority of "constitutive" secretion likely occurred via basolateral secretion and, while there is no data, such secretion likely involved low-molecular multimers.  


            The reader is advised to consult an earlier review for detail on the structure of factor VIII and its interaction with vWF (19).  Suffice to say, factor VIII is a large protein (≥ 250 kDa) whichi is physically associated with a larger protein, vWF, in the circulation. There is some evidence to suggest that factor VIII is associated with low molecular weight multimers of vWF (20).  The vWF multimer pattern in Hemofil M is different from Monoclate, another factor VIII concentrate prepared from plasma by immunoaffinity chromatography.   Hemofil M is purified using an antibody to factor VIII protein (21,22) while Monoclate is purified using an antibody to vWF (22,23).  The reasoning here is that Hemofil M will contain only vWF that is associated with it normally in the circulation while Monoclate would contain a collection of vWF with some of the unique vWF that binds to factor VIII.  The unique combination of factor VIII and vWF in  Hemofil M suggest coexpression of factor VIII and vWF resulting in secretion of a factor VIII-vWF complex  as suggested earlier by Haberichter and coworkers (24) and somewhat later by  Terraube and coworkers (25).  There are other investigators who have suggested coexpresssion and I have picked these as notable examples.  


            The elucidation of the site of synthesis of factor VIII protein has been a saga beginning more than 50 years ago 26-29).   Recent work has identified the endothelial cell as the site of factor VIII synthesis (30-33) which does provide some insight into confusion regarding organ site of synthesis in earlier studies (26-29).   The endothelial cell is also the site of vWF synthesis and it is not clear as to whether there is coexpression of factor VIII and vWF as a complex or whether secretion occurs as separate proteins which combine in the circulation. My sense is  that there is coexpression of Factor VIII with a unique vWF population to accommodate the analytical results of Fricke and Yu (20).  


            The potential basolateral secretion of proteins into the interstitial space has been somewhat neglected.  The apical or  luminal surface of the endothelial cells provides a pathway to the vascular space while the basolateral or subluminal surface provides a pathway for secretion to the interstitial space.   Basolateral secretion of fibrinogen has been demonstrated in pulmonary epithelium (34) and for  vWF in endothelial cells (10,11). The possibility of basolateral secretion of factor VIII has not been demonstrated in native endothelial cells but is suggested from the secretion of factor VIII in retroviral transduced gut epithelial cells (35).    van den Bigglelaer and coworkers (36) transduced bovine outgrowth endothelial cells (a type of circulating endothelia progenitor cell) with a lentivirus endcoding GFP-factor VIII (a chimer of green fluorescent protein and B-domain deleted factor VIII).  These investigators were able to show that the majority of the factor VIII was expressed in a constitutive manner with some 20% being taken into Weible-Palade bodies. 

The above observations on the synthesis of factor VIII in endothelial cells combined with the observations of Shovlin and coworkers (37) on expression of factor VIII in pulmonary endothelial cells show that endothelial cells have the potential to secrete factor VIII into the interstitial space via basolateral secretion.  However, we are left with a void in the understanding of the normal function of factor VIII in the interstitial space.  It is possible that dysregulation of factor VIII secretion by endothelial  could predispose to the development of fibrosis.

Conclusion:  Factor VIII is present in the interstitial space and may be involved in the development of interstitial fibrosis.

 

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