Human group II secretory phospholipase A2 (sPLA2) is an enzyme found in the alpha granules of platelets and at inflammatory sites. Although its physiological function is unclear, sPLA2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA2 activities, we developed a total chemical synthesis of sPLA2 by chemical ligation of large unprotected peptides. The synthetic segments PLA2-(1-58)-alphaCOSCH2COOH and PLA2-(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly58 and Cys59. The 124-residue polypeptide product (mass: 13,920 +/- 2 Da) was folded to yield one major product (mass: 13,905 +/- 1 Da), the loss of 15 +/- 3 Da reflecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA2 showed alpha-helix, beta-structure, and random coil contents consistent with those found in the crystal structure of sPLA2. Synthetic sPLA2 had kcat and Km values identical to those of recombinant sPLA2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA2, like recombinant sPLA2, inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca2+, and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca2+. Thus, synthetic sPLA2 is a phospholipid-independent anticoagulant like recombinant or natural sPLA2. This study demonstrates that chemical synthesis of sPLA2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA2 analogs for structure-activity studies of anticoagulant, lipolytic, or inflammatory activities.