Dennis C. Lynch

The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element

The retinoblastoma susceptibility gene product, Rb, is suspected to suppress cell growth. Rb is a 110-114 kd nuclear phosphoprotein. We have previously demonstrated that SV40 T antigen binds only to unphosphorylated Rb, and not pp112-114Rb, the family of phosphorylated Rb. Here we demonstrate the cell cycle-dependent phosphorylation of Rb. In G0/G1 cells, virtually all the Rb is unphosphorylated. In contrast, during S and G2, it is largely, if not exclusively, phosphorylated. Rb phosphorylation occurs at the G1/S boundary in several cell types tested. A 14 residue peptide, corresponding to the SV40 T domain required for transformation, is able to compete effectively with SV40 T for binding to p110Rb. We propose a model to explain how Rb may suppress cell growth by acting as a cell cycle regulatory element.

VON WILLEBRAND FACTOR, NOW CLONED

Von Willebrand factor (vWf) was, for many years, a somewhat mysterious component of the haemostatic system. In the early 1970s several groups succeeded in purifying a protein which corrected the clotting defect in haemophilic plasma (Legaz et al, 1973: Shapiro et al, 19 73). However, when antibodies raised against this ‘antihaemophilic factor’ became available, most individuals with classically defined haemophilia turned out to have normal levels of this protein (Zimmerman et al., 1971). Over the succeeding several years it became apparent that the initial preparations consisted of a high affinity complex between vWf and factor VIII (the antihaemophilic factor) (Weiss & Hoyer, 1973; Zimmerman et al, 1983). This complex was > 99% vWf by mass and, aside from correction of the clotting defect, all the properties attributed to this ‘antihaemophilic factor,’ were those of vWf. In this interval a number of inappropriate terms for vWf, such as factor VIII-related antigen, were introduced into the literature, and still persist in some textbooks. It is now clear that factor VIII and vWf are wholly different molecules with distinct physical properties. For both, molecular cloning has now been accomplished and amino acid sequences of each are available. These studies and others have helped to identify increasingly well the respective roles of factor VIII and vWf in haemostasis. vWF is active in primary haemostasis (Weiss et al, 1974): indeed, it mediates the attachment of platelets at sites of vascular injury by interacting, on the one hand, with components of the vascular wall, including collagen (Morton et al, 1983) and other undefined structures (Fauvel et al. 1983; Wagner et al, 1984), and, on the other hand, with the plateIet membrane glycoprotein Ib (Coller et al., 1983; Ruggeri et al, 1983). vWF also binds to the platelet glycoprotein IIb/IIIa complex (Ruggeri e t al, 1982. 1983), which serves as a receptor for adhesive proteins, like fibrinogen. vWF may play a role in stabilizing the initial platelet plug. vWf also prolongs the plasma half life of factor VIII, by virtue of the noncovalent but strong binding between the two proteins (Weiss et al, 1977). This role of vWf explains the decreased factor VIII activity in patients severely deficient in vWF; and conversely, the exaggerated increase of factor VIII levels seen after infusion of cryoprecipitate into these patients. vWf circulates in plasma as a set of multimers, ranging from dimers of the 2 50 000 molecular weight subunit to very large structures containing upwards of 50 subunits (Hoyer & Shainoff, 1980: Ruggeri & Zimmerman, 1980). Several lines of evidence indicate that the largest multimers are most important for the intrinsic haemostatic role of vWf. On the other hand, multimers of all sizes appear to be effective in binding factor VIII (Davies et al, 1981). vWf is synthesized in megakaryocytes (Nachman et al, 1975), which are presumably

An explanation for minor multimer species in endothelial cell-synthesized von Willebrand factor.

Initial synthesis of von Willebrand factor (vWf) by cultured human endothelial cells proceeds by formation of a dimer of pro-vWf subunits. These subunits are found only within the cell and have an apparent molecular weight of 240,000-260,000, as measured by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Posttranslational modifications, including proteolytic cleavage, glycosylation, and sulfation, result in the appearance of two additional vWf subunits. The major one migrates with the subunit of plasma vWf at an apparent molecular weight of 220,000-225,000 and the other migrates more slowly than pro-vWf at an apparent molecular weight of 260,000-275,000. These subunits oligomerize to form a set of vWf multimers, which are subsequently secreted into the culture medium. We isolated individual vWf oligomer species from the agarose gel bands and show that vWf minor, or satellite, species differ from major species in subunit composition.

The Fine Structure of von Willebrand Factor Multimers and Type IIA von Willebrand Diseasea

It is now accepted that in order for von Willebrand factor (vWf) to fulfill its biologic role, vWf subunits must be assembled into a complex multimer structure. The assembled protein must then be made available for activity at its physiologic sites: plasma, platelet alpha granules, and endothelial cells (EC). Since cultured EC synthesize, store, and secrete biologically active vWf, and EC in vivo appear to supply plasma vWf,’ cultured EC have been used to examine some of the details of these processes since 1973, when synthesis of vWf in cultured human EC was first demonstrated by Jaffe et aL2 More recent work (e.g., Refs. 3-7), using metabolic labeling, immunoisolation, and gel electrophoresis techniques, has described many of the details of EC vWf biosynthetic processing. In this presentation, I would like to rely primarily on data generated from EC to review normal von Willebrand factor multimeric structure and the subunit components that comprise the multimers. I will then discuss what changes are present in type IIA von Willebrand disease (vWd) and the mechanism by which the changes are produced, using the phenotype of a particular family as an example.

The Molecular Biology of Endothelial Cell von Willebrand Factor

von Willebrand factor, or vWf, is an adhesive plasma glycoprotein which mediates platelet attachment to areas of endothelial damage and, also, serves as a carrier for factor VIIIC, the antihemophilic factor. In the plasma, vWf is composed of a single subunit of apparent molecular weight =225 000 which is found in discrete, disulfide bonded structures ranging in size from dimers to polymers containing more than 50 subunits. Qualitative or quantitative deficencies in vWf are associated with the bleeding disorder von Willebrand’s disease, which is typified by a prolonged bleeding time in the presence of normal platelet function. In Type I vWd there is a quantitative decrease in levels of plasma vWf. In Type II vWd, of which an increasing number of subvariants have been reported, there is a qualitative alteration in vWf structure, with preferential loss of the largest multimers. Information from various studies suggests that the largest multimers are most important for the intrinsic hemostatic role of vWf, while multimers of all sizes can serve as carriers for factor VIIIC