Victoria J. Christiansen

Phase II Trial of Single Agent Val-boroPro (Talabostat) Inhibiting Fibroblast Activation Protein in Patients with Metastatic Colorectal Cancer

Purpose: Fibroblast Activation Protein (FAP) is a tumor fibroblast protease that has been shown to potentiate colorectal cancer growth. The clinical impact of FAP inhibition was tested using Val-boroPro (Talabostat), the first clinical inhibitor of FAP enzymatic activity, in a phase II study of patients with metastatic colorectal cancer. Methods: Patients with metastatic colorectal cancer who had previously received systemic chemotherapies were treated with single agent Val-boroPro 200 μg p.o. BID continuously. Eligibility included measurable disease, performance status of 0 to 2, and adequate organ function. Laboratory correlates evaluated the pharmacodynamic effects of Val-boroPro on FAP enzymatic function in the peripheral blood. Results: Twenty-eight patients (median age 62; 12 males, 16 females) were enrolled in this study. There were no objective responses. Six of 28 (21%) patients had stable disease for a median of 25 weeks (range 11-38 weeks). Laboratory analysis demonstrated significant, although incomplete inhibition of FAP enzymatic activity in the peripheral blood. Conclusion: This phase II trial of Val-boroPro demonstrated minimal clinical activity in patients with previously treated metastatic colorectal cancer. However it provides the initial proof-of-concept that physiologic inhibition of FAP activity can be accomplished in patients with colorectal cancer, and lays the groundwork for future studies targeting the tumor stroma.

Evidence that α2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation

Summary.  Background: Plasma alpha2‐antiplasmin (α2AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital α2AP deficiency results in a severe hemorrhagic disorder due to accelerated fibrinolysis. It is well established that in the presence of thrombin‐activated factor XIII (FXIIIa), α2AP becomes covalently ligated to the distal α chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that α2AP is covalently linked to plasma fibrinogen . That singular observation led to our hypothesis that native plasma factor XIII (FXIII), which is known to catalyze covalent cross‐linking of fibrinogen in the presence of calcium ions, can also incorporate α2AP into fibrinogen in the circulation. Results and Conclusions: We now provide evidence that FXIII incorporates I125‐labelled α2AP into the Aα‐chain sites on fibrinogen or fibrin. We also measured the content of α2AP in isolated plasma fibrinogen fractions by ELISA and found that substantial amounts were present (1.2–1.8 moles per mole fibrinogen). We propose that α2AP becomes ligated to fibrinogen while in the circulation through the action of FXIII, and that its immediate presence in plasma fibrinogen contributes to regulation of in vivo fibrinolysis.

Complement C5b-9 Increases Plasminogen Binding and Activation on Human Endothelial Cells

Deposition of the terminal complement proteins (C5b-9) on human endothelial cells can result in cell lysis or nonlytic alterations of cell function including procoagulant responses. Because regulation of fibrinolysis is a central endothelial function and because C9 contains a carboxyl-terminal lysine similar to other proteins that bind and facilitate activation of plasminogen (PG), the effects of complement injury on PG binding and activation on these cells were investigated. Activation of complement through deposition of C5b67 complexes on endothelial cells resulted in a small increase (approximately 20%) in PG binding. Incorporation of C8 into C5b-8 resulted in no further increase in binding; however, specific 125I-PG binding was increased by approximately 100% after C5b-9 deposition. Moreover, PG was found to bind specifically to C7 and C9. The PG bound to endothelial cells after C5b-9 deposition was readily activated by tissue-type plasminogen activator (TPA). In a cell-free system, complement C9 and a synthetic peptide composed of the 20 carboxyl-terminal amino acids of C9 enhanced PG activation by TPA. Removal of the carboxyl-terminal lysine of C9 abolished the enhancement of PG activation without diminishing PG binding. We conclude that membrane C9 may comprise a binding site for PG and serve to enhance activation of this zymogen by TPA. These findings suggest that immune injury to the endothelium may enhance both the fibrin-generating and fibrinolytic capacity of the vessel wall.

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin

Summary.  Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α2‐antiplasmin (α2AP) incorporated within fibrin. Circulating prolyl‐specific serine proteinase, antiplasmin‐cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met‐α2AP between ‐Pro12‐Asn13‐ to yield Asn‐α2AP, which is crosslinked to fibrin approximately 13× more rapidly than Met‐α2AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met‐α2AP to Asn‐α2AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl‐Arg‐(8‐amino‐3,6‐dioxaoctanoic acid)‐d‐Ala‐l‐boroPro selectively inhibited APCE vs. DPPIV, with an apparent Ki of 5.7 nm vs. 6.1 μm, indicating that an approximately 1000‐fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent Ki of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose‐dependent decrease of APCE‐mediated Met‐α2AP cleavage, which ultimately shortened plasminogen activator‐induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC50 value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.

Angiotensin-converting enzyme gene polymorphism in a cohort of coronary angiography patients

An association between a polymorphism of the angiotensin-converting enzyme (ACE) gene and myocardial infarction (MI) in men has been previously reported. The present study examines the association between ACE genotype, atherosclerosis, MI, hypertension and other cardiovascular risk factors in Caucasian men (n=576) and women (n=124) who have undergone coronary angiography. Gene frequencies are also reported for African-American men (n=56). Genotype determination was based on the presence (allele I) or absence (allele D) of a 287 nucleotide Alu sequence in intron 16 of the ACE gene. Genotype frequencies for DD, ID and II were: 30.9, 47.7, 21.4% for Caucasian men; 28.2, 48.4, 23.4% for Caucasian women; and 30.4, 46.4, 23.2% for African-American men. There were no statistically significant associations between ACE genotype and number of plaques (> or =10% obstruction), lipid variables, or body mass index (BMI) for Caucasian men. Caucasian women with the DD genotype had on average fewer plaques, but this was accounted for by their younger ages. In Caucasian males, the DD genotype independently contributed to the presence of hypertension (odds ratio=1.8, 95% CI 1.1-2.9) after adjusting for age and BMI. In Caucasian males with total cholesterol levels less than 200 mg/dl (n=237), the DD (odds ratio=2.5, 95% CI 1.2-5.4) and ID genotypes (odds ratio=2.2, 95% CI 1.1-4.4) were associated with a history of MI.

Crosslinking of α2‐Antiplasmin to Fibrin

Abstract: Human α2‐antiplasmin (α2AP) is the primary inhibitor of plasmin‐mediated fibrinolysis and is an efficient substrate of activated factor XIII (FXI‐IIa). Among 452 amino acid residues in α2AP, Gln2 is believed to be the sole FXI‐IIa‐reactive site that participates in crosslinking α2AP to fibrin. We studied the effect of mutating Gln2 on the ability of FXIIIa to catalyze crosslinking of α2AP to fibrin. By FXIIIa catalysis, [14C]methylamine was incorporated into a Q2A‐α2AP mutant in which Gln2 (Q) was replaced by Ala (A), thereby indicating that wildtype α2AP has more than one FXIIIa‐reactive site. To identify the FXIIIa‐reactive sites in α2AP, wildtype α2AP and Q2A‐α2AP were labeled with 5‐(biotinamido)pentylamine by FXIIIa. Each labeled α2AP was digested with trypsin and applied to an avidin affinity column to capture labeled peptides. Edman sequencing and mass analysis of each labeled peptide showed that out of 35 Gln residues in wildtype α2AP, four were labeled with the following order of efficiency: Gln2 > Gln21 > Gln419 > Gln447. Q2A‐α2AP was also labeled at the three minor sites, Gln21 > Gln419 > Gln447. Q2A‐α2AP became crosslinked to fibirin(ogen) by FXIIIa catalysis at approximately one‐tenth the rate of wt‐α2AP. These results demonstrate that α2AP has one primary (Gln2) and three minor substrate sites for FXIIIa and that the three minor sites identified in this study can also participate in crosslink formation between α2AP and fibrin, but at a much lower efficiency than the Gln2 site.

Why α2-antiplasmin must be converted to a derivative form for optimal function

Summary.  Background: Human α2‐antiplasmin (α2AP), the primary inhibitor of fibrinolysis, is secreted from the liver into plasma as a 464‐residue protein with Met as the N‐terminus. An R6W polymorphism has been suggested to affect fibrinolytic rate. Within circulating blood, antiplasmin‐cleaving enzyme (APCE) cleaves Met‐α2AP(R6) faster than Met‐α2AP(W6) at the Pro12–Asn13 bond to yield Asn‐α2AP. Objectives: To compare Met‐α2AP(R6), Met‐α2AP(W6) and Asn‐α2AP for crosslinking with fibrin and the ability to protect fibrin from digestion by plasmin. Methods and results: Asn‐α2AP utilizes Gln2 (Gln14 in Met‐α2AP) to become crosslinked to fibrin approximately twelvefold faster than Met‐α2AP(R6) or Met‐α2AP(W6), and this enhances the resistance of fibrin to plasmin. All three forms of α2AP inhibit plasmin at identical rates. The N‐terminal 12‐residue peptide of Met‐α2AP slows crosslinking of Met‐α2AP(R6) or Met‐α2AP(W6) by limiting access of factor XIIIa to Gln14 rather than shifting crosslinking to other Gln residues. Edman sequencing and mass analyses of tryptic peptides from each α2AP crosslinked with 5‐(biotinamido)pentylamine showed Gln14 as the only major crosslinking site. Residues 5–8, GRQL in Met‐α2AP(R6), and residues 1–8, MEPLGWQL in Met‐α2AP(W6), slow fibrin crosslinking. Conclusion: Gln14 in both Met‐α2AP(R6) and Met‐α2AP(W6) is sheltered by the N‐terminal 12‐residue peptide, which, when cleaved, yields Asn‐α2AP, which is rapidly crosslinked to fibrin and maximally protects it from plasmin. The R6 W polymorphism in Met‐α2AP does not affect its crosslinking to fibrin, but it does slow cleavage by APCE and reduces the amount of Asn‐α2AP available for rapid crosslinking to fibrin.

Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and removal.

Alpha2-antiplasmin (alpha2AP) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Two forms of alpha2AP circulate in human plasma: a 464-residue protein with methionine as the amino-terminus (Met-alpha2AP) and an N-terminally-shortened 452-residue form with asparagine as the amino-terminus (Asn-alpha2AP). Human plasma alpha2AP concentration is 1 micro M and consists of approximately 30% Met-alpha2AP and approximately 70% Asn-alpha2AP. The major form (Asn-alpha2AP) is rapidly crosslinked to fibrin during blood clotting by activated coagulation factor XIII and as a consequence, fibrin becomes more resistant to fibrinolysis. It is apparent that alpha2AP is important in modulating the effectiveness and persistence of fibrin with respect to its susceptibility to digestion and removal by plasmin. Hence, the physiologic role of alpha2AP suggests that it may be a useful target for developing more effective treatment of thrombotic diseases. Research on alpha2AP appears to be moving in two main directions: (1) efforts to use variant forms of alpha2AP to reduce bleeding secondary to thrombolytic therapy while not slowing thrombolysis; and (2) efforts to use variant forms to diminish the activity of alpha2AP as a plasmin inhibitor so that fibrinolysis becomes enhanced. Methods to accomplish these two goals mostly involve manipulation of defined functional domains within the molecular structure of alpha2AP, or inhibition of a newly described novel plasma proteinase, termed antiplasmin-cleaving enzyme, that generates the more favorable form of alpha2AP, Asn-alpha2AP, for crosslinking to fibrin. The antiplasmin-cleaving enzyme has similarity in primary structure and catalytic properties to fibroblast activation protein/seprase. This review summarizes recent studies that may hold promise for modulating alpha2AP activity and its interactions with certain proteins as new therapeutic strategies for preventing and treating thrombotic disorders.

Protein kinase C-ζ activity but not level is decreased in Alzheimer's disease microvessels

While there is considerable evidence demonstrating altered activity of the major isoforms of protein kinase C (PKC) in the vasculature and neurons of Alzheimer disease (AD) brains, little is known about the activity and/or levels of the atypical PKC isoforms. The objective of this study is to compare PKC-zeta activity and level in cerebral microvessels isolated from AD brains with microvessels from the brains of nondemented age-matched controls. Measurements of the kinase activity reveals that the PKC-zeta activity is significantly (P < 0.01) lower in AD brain microvessels compared with vessels from control brain. Despite this decrease in enzyme activity, the level of PKC-zeta, assessed by Western blot, is significantly (P < 0.01) elevated in AD microvessels. These data demonstrate significant and divergent changes in the PKC-zeta activity and level in the microcirculation of the AD brain and suggest that aberrant regulation of microvascular PKC-zeta could contribute to the abnormal signaling mechanisms at the blood-brain barrier in the AD brain.

Suppression of tumor growth in mice by rationally designed pseudopeptide inhibitors of fibroblast activation protein and prolyl oligopeptidase.

Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth > 90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs.