Big piece, little piece or: Yes, factor VIII is a protein

Abstract

Thiswillbelessahistoricalsketch,betterlefttohistorians,thana memoir and an approximation. Notwithstanding 35 yearsandcounting, IwasaBiochemistrygraduatestudentatChapelHill and knew next to no one in the wider circles of hemostasisresearch beyond our campus. The world was substantiallylarger andmoreexpensivethen;graduatestudents did notearnas much as they do now. Vietnam was also very much on ourminds. At 23, I was naive about the culture of science and theways that personal temperament and style influence theproduction of a line of inquiry. This makes it all the moreinteresting to look back, if a little daunting to fill in details, asmemoryofatimeiscoloredbyone’sviewpointatthetime.Theelucidationoffactor(F)VIIIstructureanditsdevelopmentintomodern therapies for hemophilia A has been reviewedthoroughly by Tuddenham [1]. I will fill in some of the run-up to that story from the perspective of a 3-year experience inthe early light of the transformation of FVIII into a molecularentity.The affirmative offered by my title answers the cognatequestion in the title of a paper by Veder [2] published in Naturejust 1 year before I enrolled in graduate school at theUniversity of North Carolina in Chapel Hill, and 2 yearsbefore I started in earnest on the biochemistry of FVIII. Therewas ample circumstantial evidence, and not a little conviction,that it was a protein, as FVIII coagulant was known to beimmunogenic, including as a complication of therapeutictransfusion [3,4]. The unequivocal answer came more than12 yearslater[5],butinthemeantimeIhadthegoodfortunetolandrightinthemidstofawatershedinthequestforthenatureof FVIII. My mentor Robert Wagner was an intense but quietand introspective biochemist who did not stir us up much withanxiety over the competition; we in the lab were a relativelysmall family of students and staff, not insular, but concernedmorewiththetaskathandthanwiththeactionabroad.Unnoticed by me, therewas ample action: the year I graduated(January1972)markedthebeginningofthedecisiveprocessionof papers on the structure and genetics of FVIII.Oneofthemostrousingaspectsofscienceisthatparanoiaisredundant, because everyone is out to get you. This is whatmakes progress possible, after all. (Max Planck)In the late 1960s, the solution to the biochemistry andgenetics of FVIII, hemophilia A and von Willebrand disease(VWD) was at hand. Three essential features had beenestablished: (i) FVIIIc (coagulant) is associated with anexceptionallylargemacromolecule;(ii)FVIIIcbecomessmallerwhen the ionic strength is raised; (iii) hemophilic plasmacomplements VWD in vivo after transfusion, but not afteraddition to blood in vitro. With the one-gene–one-proteinconcept firmly established, it was a small leap in logic to relatethe genetics of the two FVIII hemophilias to a carrier–cargotype of biochemistry for autosomal von Willebrand factor(VWF) and X-linked FVIII coagulant. In retrospect, thestructuraldetails, whileinteresting,werenotallthatimportant.Yet, the birth of the idea was contentious and, even within thecorridors of McNider Hall on the Chapel Hill campus, debatewas spirited.The information essential to define VWF as a protein, itsfunction as a carrier of FVIII and the solution to the enigma ofVWD began with a paper by Wagner and graduate studentMurray Thelin [6], himself a hemophiliac. They observed thatsimply raising the ionic strength of plasma or FVIII concen-trates caused a sharp decrease in the sedimentation coefficientand, by inference, the molecular weight of FVIII coagulantactivity.Thiswasnosmallfeat.PlasmaandFVIIIconcentrateswere subjected to ultracentrifugation in a preparative ultracen-trifuge (among the first ultracentrifuges sold: Spinco Model L,serial number 012, SW40 rotor no. 007, I recall) for a timesufficient to sediment FVIIIc activity part-way down the 5-mL(swing-out) tube. FVIIIc could be identified only with theactivated partial thromboplastin time (APTT)-based clottingassay. To sample the centrifuge tube, they bent the tip of ahypodermic needle into a J and mounted it J-tip up on a rackand pinion, so when the needle was lowered an increment at atime,smallsamplesfromthemeniscusdowncouldbeaspiratedintoamicroburetteandassayed.Thetime-dependentinflectionpoint in FVIII activity between the depleted upper andundepleted lower regions of the tube was used to calculate thesedimentationcoefficient,whichwas21SinaFVIIIconcen-trate. This is greater than that of the small (18S) ribosome sub-unit.FVIIIbecame4.3SabruptlywhentheNaClconcentration