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Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substance(s) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor Vlla and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXadependent inhibition of fVIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

Site-specific substitutions of arginine for lysine in the thermostable D-xylose isomerase (XI) from Actinoplanes missouriensis are shown to impart significant heat stability enhancement in the presence of sugar substrates most probably by interfering with nonenzymatic glycation. The same substitutions are also found to increase heat stability in the absence of any sugar derivatives, where a mechanism based on prevention of glycation can no longer be invoked. This rather conservative substitution is moreover shown to improve thermostability in two other structurally unrelated proteins, human copper, zinc-superoxide dismutase (CuZnSOD) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The stabilizing effect of Lys----Arg substitutions is rationalized on the basis of a detailed analysis of the crystal structures of wild-type XI and of engineered variants with Lys----Arg substitution at four distinct locations, residues 253, 309, 319, and 323. Molecular model building analysis of the structures of wild-type and mutant CuZnSOD (K9R) and GAPDH (G281K and G281R) is used to explain the observed stability enhancement in these proteins. In addition to demonstrating that even thermostable proteins can lend themselves to further stability improvement, our findings provide direct evidence that arginine residues are important stabilizing elements in proteins. Moreover, the stabilizing role of electrostatic interactions, particularly between subunits in oligomeric proteins, is documented.

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Summary. The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen (mPlg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin (mPli) and equilibrated with 5 mmol L À1 citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg À1 mPli reduced infarct size at 24 h from 27 (26-30) to 25 (21-28) mm 3 (median and range, n ¼ 16 each, P ¼ 0.0001), whereas 4.0 mg kg À1 rt-PA and 40 mg kg À1 mPlg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14-30) to 9.1 (3.1-25) mm 3 with 5.0 mg kg À1 mPli (n ¼ 15 each, P <0.002) and to 11 (5.2-27) mm 3 with 4.0 mg kg À1 rt-PA (n ¼ 6; P ¼ 0.02). Infarct reduction was still observed at 10 h after occlusion with mPli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg À1 mPli over 2 h, induced 51 AE 15% lysis (mean AE SD, n ¼ 11) vs. a control value of 23 AE 5.5%. mPli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of mPli in both models was similar to that of intact plasmin. These findings indicate that recombinant mPli may be useful for treatment of ischemic stroke and arterial thrombosis.