At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis suggested to us that a thrombin variant that lacked procoagulant activity while retaining anticoagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose-dependent, reversible anticoagulation without any indication of procoagulant activity. Notably, template bleeding times were not prolonged, suggesting a reduced potential for bleeding complications.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

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Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its antiproliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) ␦ and ⑀ and found it to be a competitive inhibitor of both these enzymes. The apparent K i values for PMEGpp were 3-4 times lower than the K m values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol ␦ and pol ⑀ to repair the incorporated PMEG revealed that pol ⑀ could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control templateprimers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3-5-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol ⑀, pol ␦ exhibited negligible activity on these template-primers, indicating that pol ⑀, but not pol ␦, can repair the incorporated analog. PMEG1 (9-(2-phosphonylmethoxyethyl)guanine) ( Fig. 1) is a member of a new class of acyclic nucleotide analogs characterized as phosphonylmethyl ethers. Several analogs in this class have demonstrated broad spectrum antiviral activity (1). PMEG is being evaluated as an antitumor agent for cancer treatment. Its anti-proliferative activity has been demonstrated in vitro against human leukemic cells (2) as well as a number of solid tumor cell lines (3). PMEG also showed antitumor activity in two types of mouse transplantable tumors, P388 lymphocytic leukemia and B16 melanoma (4). In addition, the analog suppressed the growth of papillomavirus-induced condylomas on human foreskin xenografts in mice (5).Once inside the cells, cellular enzymes phosphorylate the analog to PMEG mono-and diphosphate (6). The diphosphate form (PMEGpp) is an analog of dGTP. Although the mechanism of action of PMEG has not yet been determined, PMEG diphosphate has been shown to be a potent inhibitor of rat DNA polymerase ␣ and ⑀ (7) and to be incorporated into DNA in vitro by human DNA polymerase ␣ and ␦ (8, 9). Therefore, it can potentially inhibit cellular DNA synthesis.DNA polymerases ␣, ␦, and ⑀ are the enzymes cooperating in chromosomal DNA replication (10). Pol ␣, associated with DNA primase activity, synthesizes RNA-DNA primers for initiation of DNA replication at ori sites and for priming of Okazaki fragments on the lagging strand of DNA. Pol ␦, in cooperation with proliferating cell nuclear antigen (PCNA) and other protein factors, synthesizes the leading strand of DNA. Pol ⑀, which is dispensable for SV40 DNA replication (11) but not for cellular DNA replication (12), may be required as a second DNA polymerase on the lagging DNA strand (13). In contrast to pol ␣, both pol ␦ and pol ⑀ have intrinsic 3Ј-5Ј-exonuclease activity associated with a proofreading function (14). Both enzymes are necessary for the repair of cellular DNA damage after UV ...