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BSAP has been identified previously as a transcription factor that is expressed at early, but not late, stages of B-cell differentiation. Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins. BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse. An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP. Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1. During embryogenesis, the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system (CNS). Later, the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis. BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse. All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis.

A chimeric protein was produced with the N-terminal domain (amino acids 1-45) of annexin I and the core of annexin V (amino acids 19-320). This protein, annexin IN-VC, has a similar Ca2+ requirement for binding to phospholipid bilayers of 20% phosphatidylserine (PS)/80% phosphatidylcholine (PC) as annexin V. In contrast to annexin V, this protein has a strong potency to aggregate phospholipid vesicles as is shown by turbidimetric measurements and cryo-electron microscopy. Ellipsometry was employed to study quantitatively the phenomenon of phospholipid vesicle adhesion to annexin IN-VC bound to a planar phospholipid bilayer. The amount of phospholipid vesicles bound by annexin IN-VC on the planar bilayer is proportional to its surface coverage and can be inhibited by coadsorption of annexin V on the planar bilayer or by shielding the phospholipid surface of the vesicles with blood coagulation factor Va. Annexin IN-VC, like annexin V, does not bind to pure PC bilayers, but its adsorption on anionic phospholipid bilayers brings about the capacity to bind pure PC vesicles. This suggests that annexin IN-VC generates or exposes after binding to anionic phospholipids another phospholipid binding site, that differs from the annexin V phospholipid binding site. Collectively, the data suggest that two-dimensional cluster formation of annexin IN-VC on a bilayer with anionic phospholipids is involved in vesicle adherence.

Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Eschevichia cofi expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.Blood coagulation comprises a complex cascade of enzymatic conversions of zymogens into activated proteases finally resulting in clot formation. Physiologically relevant rates of conversion occur when enzyme and substrate interact with specific protein cofactors and surfaces and thus form a complex (reviewed in [l] Recently a novel anticoagulant was isolated from human umbilical cord arteries, which inhibits thromboplastin as well as factor-Xa-induced clotting but does not affect thrombininitiated fibrin formation [6]. This vascular anticoagulant (VAC) has been shown to inhibit phospholipid-dependent procoagulant reactions by high-affinity binding to phospholipids thereby inhibiting their catalytic activity in these reactions [7]. Hence VAC attacks the enzyme complex at a site which differs from the ones described above. This paper reports the molecular cloning of the cDNA of human VAC and its expression in Escherichia coli. The recombinant VAC possesses an anticoagulant activity indistinguishable from its natural counterpart. MATERIALS AND METHODS Sequence analysis of tryptic peptidesThe 32-kDa vascular anticoagulant (VAC) was isolated from human umbilical cord arteries and subsequently from Correspondence to R. Hauptmann, Ernst Boehringer Institut fur Arzneimittelforschung, Dr. Boehringer-Gasse 5/11, A-I 121 Wien, AustriaAhhreviutions. VAC, vascular anticoagulant; bp, base pairs; DPhe-Pip-Arg-NH-Np ' 2 HCI (S2238), D-phenylalanyl-L-pipecolyl-Larginine p-nitroanilide dihydrochloride ; OleZGroPCho, 1,2-dioleoylsn-gl ycero-3-phosphocholine ; Ole2GroPSer, 1,2-dioleoyl-sn-glycero-3-phosphoserine. PFP, platelet-free plasma.___-placenta [6, 81. The two proteins turned out to be identical. An additional purification step was performed by reversephase HPLC on a Bakerbond WP c 1 8 column (4.6 x 250 mm, 5 pm particle diameter, 30 nm pore diameter) using a 24-min gradient of 20-68% acetonitrile in 0.1 % trifluoroacetic acid in water. The flow rate was 1 ml/min, detection was done by ultraviolet absorption at 214 nm and 280 nm simultaneously. The main peak was collected and dried in a Speed Vac concentrator. An aliquot of the residue was applied to an SDS gel to prove its identity with the 32-kDa vascular anticoagulant.VAC was dissolved in 1% ammonium bicarbonate at 1 mg/ml. Trypsin (Worthington tosylphenylalanylchloromethane-treated) was added to 2% (by mass) and the solution incubated at 37 "C for 6 h. After a second addition of 2% (by mass) trypsin the incubation was continued overnight. The digestion was terminated by freezing. Tryptic peptides were separated by reverse-phase HPLC on a Waters p-Bondapak C...

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