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A variety of plasma-derived (pd) and recombinant (r) factor VIII (FVIII) concentrates are used to prevent and treat bleeding in severe hemophilia A patients. A significant side effect of FVIII replacement is the development of FVIII neutralizing antibodies (inhibitors) in up to 30% of patients receiving FVIII concentrates. The FVIII protein content (FVIII:Ag) per unit of FVIII:C in FVIII concentrates, and how effectively the FVIII:Ag in FVIII concentrates binds to von Willebrand factor (VWF) may provide information relevant for the survival of FVIII:C in vivo and for estimating the risk for inhibitor development. The FVIII:Ag content of nine r-FVIII and nine pd-FVIII concentrates were quantified in this study using two enzyme-linked immunosorbent assay (ELISA) platforms. The two ELISA platforms were based on the use of a monoclonal anti-(FVIII light chain)-IgG and polyclonal anti-FVIII antibodies as capture antibodies and both ELISAs were equally able to detect > or =0.005 IU of FVIII:Ag. Measured in international units, the r-FVIII concentrates contained significantly higher FVIII:Ag per unit of FVIII:C than the pd-FVIII concentrates. The VWF-binding profiles of the r-FVIII and pd-FVIII concentrates were also determined by gel filtration chromatography. Unlike the plasma-derived products, the r-FVIII concentrates invariably contained a fraction of FVIII:Ag molecules (approximately 20%) which was unable to associate with VWF. Given that VWF regulates both factor VIII proteolysis and survival of FVIII:Ag in vivo, the fraction of FVIII:Ag unable to bind to VWF may have a reduced survival and be more susceptible to proteolytic degradation in vivo. The extent to which the fractions of FVIII:Ag in concentrates able and unable to bind to VWF contribute to inhibitor development in severe FVIII-deficient patients is unknown.

We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1-41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after <1.0 nM ␣-thrombin, 10 M SFLLRN, or 100 M AYPGKF were added to platelets. When SFLLRN or AYGPKF were added to platelets, generation of PAR-1 and -4 activation peptides was complete at 10 s. Generation of both PAR-1 and -4 activation peptides in response to 1 nM ␣-throm- The function of protease-activated receptor 1 (PAR-1) 2 in directing the responses of human platelets to various concentrations of human ␣-thrombin has been well documented (1-3). Activation of human platelet PAR-1 arising from cleavage at Arg 41 -Ser 42 in response to ␣-thrombin and the simultaneous exposure of the previously cryptic tethered ligand domain (beginning with the sequence 42 SFLLRN), were first reported by Vu and colleagues (4). Two studies have confirmed cleavage at Arg 41 -Ser 42 by quantifying platelet PAR-1 activation peptide release (5, 6). Monoclonal antibodies directed against the hirudin-like ␣-thrombin-binding domain of PAR-1 or against residues spanning the reported ␣-thrombin cleavage site (PAR-1 span antibodies) practically eliminated all responses of human platelets to Յ1 nM ␣-thrombin. However, Ն10 nM ␣-thrombin overcame inhibition by either PAR-1 monoclonal antibody, allowing human platelets to respond normally (5, 7).A second thrombin receptor on human platelets, namely PAR-4, lacks the hirudin-like ␣-thrombin-binding domain of PAR-1, and apparently requires Ն10.0 nM ␣-thrombin for its activation and participation in human platelet activation (8 -10). Cleavage of human platelet PAR-4 at Arg 47 -Gly 48 and the release of PAR-4-(1-47) as a direct index of PAR-4 activation have not been reported. Whether Ն10 nM ␣-thrombin only activates PAR-4, or simultaneously activates PAR-1 when human platelets are activated in the presence of the monoclonal anti-PAR-1 antibodies above has not been reported (5,7,9). Dual protease-activated receptors, namely PAR-3 and -4, also govern the responses of mouse platelets to thrombin in vivo and in vitro (11-13). PAR-3 serves as a co-factor for PAR-4 activation on mouse platelets (11, 12) by altering exosite 1 of murine ␣-thrombin in a manner that promotes the diffusion of PAR-1 into the active center of murine ␣-thrombin (14).PAR-1 and -4 on human platelets are activated and signal inter-dependently. A recent study has reported that dimerization of PAR-1 and -4 on human platelets facilitates PAR-4 cleavage by ␣-thrombin (15). We have reported previously that SFLLRN propagates PAR-1 activation measured as the [PAR-1-(1-41)] that is released into activated platelet supernatants (16). The present study investigates whether PAR-1 activation also facilitates activation of PAR-4 on human platelets. Using a * This work was supported by a grant-in-aid from the Heart and S...

1. The administration of dihydrotestosterone to rats orchidectomized 7 days previously stimulated the synthesis of nuclear receptor in prostatic cells several hours in advance of DNA synthesis and mitosis. 2. The synthesis of nuclear receptor is tightly coupled to cell proliferation; consequently, in resting cells, there is no further net synthesis of nuclear receptor above the maximum of approx. 8000 molecules/cell. 3. After orchidectomy a rapid decline in the concentration of free androgen in the nuceus and a slower decline in the concentration of nuclear receptor are observed. 4. Owing to the apparent scarcity of receptor-inactivating factors in the nucleus, and the inverse relationship between amounts of nuclear and cytoplasmic receptors, it is concluded that the nuclear receptor is discharged into the cytoplasm after orchidectomy. 5. The formation of the cytoplasmic receptor is an early event preceding the onset of cellular autolysis. 6. Regressing prostate develops the capacity to eliminate cytoplasmic receptor, and this capacity is retained by the regenerating prostate for at least 14 days. 7. The synthesis of nuclear receptor in early G1 phase may control the entry of cells into the cell cycle and the prolonged retention of receptor in the nucleus may prevent the activation of autophagic processes.