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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...

A cDNA was cloned coding for a new member of the human CaZf-modulated phospholipid-binding protein family termed annexins. Due to its 56% identity to the human vascular anticoagulant (VAC) the new protein is named VAC-fi, renaming the previous VAC as VAC-a. Northern analysis detects one hybridizing mRNA species of 2.2 kb in human placenta. Genomic Southern blot analysis shows a VAC-8 gene of comparable complexity to the VAC-a gene. The cDNA was expressed in Eschcrichia coliand the recombinant protein purified to homogeneity. Antiserum raised against VAC-j weakly cross-reacts with VAC-x. The properties of VAC-j as an anticoagulant and as an inhibitor of phospholipase A2 activity were analyzed and compared to those of VAC-a.Phospholipid surfaces play an important regulatory role in blood coagulation by catalyzing certain procoagulant reactions I1 -31. Recently a protein, termed vascular anticoagulant (VAC), was discovered, which controls the catalyzing capacity of phospholipids [4,5]. VAC extends the set of anticoagulants acting on the four component procoagulant complex, consisting of either coagulation factors X, IXa, VIIIa or factors 11, Xa Cloning of the corresponding cDNA and sequence analysis showed that VAC is a member of the annexin family [7, 81 and identical