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

BACKGROUND AND OBJECTIVE: Resistance of thrombi to plasmin digestion depends primarily on the amount of α(2)-antiplasmin (α(2)AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-α(2)AP between -Pro12-Asn13- to yield Asn-α(2)AP, which is crosslinked to fibrin approximately 13× more rapidly than Met-α(2)AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-α(2)AP to Asn-α(2)AP 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 K(i) 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 K(i) 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-α(2)AP 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 IC(50) 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.

Evidence that alpha2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation.

BACKGROUND: Plasma alpha2-antiplasmin (alpha2AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital alpha2AP 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), alpha2AP becomes covalently ligated to the distal alpha chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that alpha2AP 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 alpha2AP into fibrinogen in the circulation. RESULTS AND CONCLUSIONS: We now provide evidence that FXIII incorporates I 125-labelled alpha2AP into the Aalpha-chain sites on fibrinogen or fibrin. We also measured the content of alpha2AP 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 alpha2AP 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.

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

BACKGROUND: Human alpha(2)-antiplasmin (alpha(2)AP), 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-alpha(2)AP(R6) faster than Met-alpha(2)AP(W6) at the Pro12-Asn13 bond to yield Asn-alpha(2)AP. OBJECTIVES: To compare Met-alpha(2)AP(R6), Met-alpha(2)AP(W6) and Asn-alpha(2)AP for crosslinking with fibrin and the ability to protect fibrin from digestion by plasmin. METHODS AND RESULTS: Asn-alpha(2)AP utilizes Gln2 (Gln14 in Met-alpha(2)AP) to become crosslinked to fibrin approximately twelvefold faster than Met-alpha(2)AP(R6) or Met-alpha(2)AP(W6), and this enhances the resistance of fibrin to plasmin. All three forms of alpha(2)AP inhibit plasmin at identical rates. The N-terminal 12-residue peptide of Met-alpha(2)AP slows crosslinking of Met-alpha(2)AP(R6) or Met-alpha(2)AP(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 alpha(2)AP crosslinked with 5-(biotinamido)pentylamine showed Gln14 as the only major crosslinking site. Residues 5-8, GRQL in Met-alpha(2)AP(R6), and residues 1-8, MEPLGWQL in Met-alpha(2)AP(W6), slow fibrin crosslinking. CONCLUSION: Gln14 in both Met-alpha(2)AP(R6) and Met-alpha(2)AP(W6) is sheltered by the N-terminal 12-residue peptide, which, when cleaved, yields Asn-alpha(2)AP, which is rapidly crosslinked to fibrin and maximally protects it from plasmin. The R6 W polymorphism in Met-alpha(2)AP does not affect its crosslinking to fibrin, but it does slow cleavage by APCE and reduces the amount of Asn-alpha(2)AP available for rapid crosslinking to fibrin.

Crosslinking of alpha 2-antiplasmin to fibrin.

Human alpha 2-antiplasmin (alpha 2AP) is the primary inhibitor of plasmin-mediated fibrinolysis and is an efficient substrate of activated factor XIII (FXIIIa). Among 452 amino acid residues in alpha 2AP, Gln2 is believed to be the sole FXIIIa-reactive site that participates in crosslinking alpha 2AP to fibrin. We studied the effect of mutating Gln2 on the ability of FXIIIa to catalyze crosslinking of alpha 2AP to fibrin. By FXIIIa catalysis, [14C]methylamine was incorporated into a Q2A-alpha 2AP mutant in which Gln2 (Q) was replaced by Ala (A), thereby indicating that wildtype alpha 2AP has more than one FXIIIa-reactive site. To identify the FXIIIa-reactive sites in alpha 2AP, wildtype alpha 2AP and Q2A-alpha 2AP were labeled with 5-(biotinamido)pentylamine by FXIIIa. Each labeled alpha 2AP 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 alpha 2AP, four were labeled with the following order of efficiency: Gln2 > Gln21 > Gln419 > Gln447. Q2A-alpha 2AP was also labeled at the three minor sites, Gln21 > Gln419 > Gln447. Q2A-alpha 2AP became crosslinked to fibirin(ogen) by FXIIIa catalysis at approximately one-tenth the rate of wt-alpha 2AP. These results demonstrate that alpha 2AP 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 alpha 2AP and fibrin, but at a much lower efficiency than the Gln2 site.

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.

Cross-linking of wild-type and mutant alpha 2-antiplasmins to fibrin by activated factor XIII and by a tissue transglutaminase.

Human alpha(2)-antiplasmin (alpha(2)AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in alpha(2)AP, only Gln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k(cat)/K(m)((app))) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type alpha(2)AP (WT-alpha(2)AP) and Q2A mutant alpha(2)AP (Q2A-alpha(2)AP). [(14)C]Methylamine incorporation showed the k(cat)/K(m)((app)) of FXIIIa to be 3-fold greater than that of tTG for WT-alpha(2)AP. With FXIIIa or tTG catalysis, [(14)C]methylamine was incorporated into Q2A-alpha(2)AP, indicating that WT-alpha(2)AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-alpha(2)AP or Q2A-alpha(2)AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled alpha(2)AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln(2) > Gln(21) > Gln(419) > Gln(447). Q(2)A-alpha(2)AP was also labeled at Gln(21) > Gln(419) > Gln(447), but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha(2)AP. These results show that alpha(2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in alpha(2)AP and yields the same order of reactivities.

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.

Effects of ricin on the ability of rabbit arteries to contract and relax.

Ricin, a toxic lectin from castor beans, reduces blood pressure. The current studies determined the effect of ricin on contractions in response to norepinephrine (NE) of rabbit central ear artery, endothelium-dependent relaxations to methacholine and ATP of aorta rings and endothelium-independent relaxations to papaverine in central ear artery and aorta rings. Rabbits were given 0.11 or 0.22 micrograms kg-1 ricin i.v. and 18 h, 4 days, or 7 days later the arteries were removed and tested. Maximal contractions to NE were increased by 8-23% (P > 0.05) with ricin treatment. The EC50 was increased in five of six ricin-treated groups, although only significantly so at 18 h after a minimum lethal dose of ricin. Maximum relaxations to methacholine were increased by 20-57% (P > 0.05) at 18 h following both ricin doses but returned to control values at later time points. Relaxations in response to ATP were significantly enhanced in all ricin-treated groups (95-205%) except in the 18 h and 4-day 0.11 microgram kg-1 dose groups where the increase was 0-46%. Relaxations in response to papaverine were not altered in either artery. Thus, ricin decreases the sensitivity of the rabbit central ear artery to NE and increases endothelial-dependent relaxations of the rabbit aorta. Therefore, decreased blood pressure following ricin administration may be due to vasodilation caused by decreased vascular contractions and increased endothelial-dependent vascular relaxation.

The effects of ricin on the sympathetic vascular neuroeffector system of the rabbit.

Ricin is a toxic lectin that inhibits protein synthesis. Because ricin decreases arterial pressure and causes cardiovascular collapse, its effects on the vascular neuroeffector system were investigated. Rabbits were given either of two doses of ricin, and then norepinephrine (NE) release from aorta to transmural stimulation, NE uptake into aorta, NE content of aorta, monoamine oxidase activity, and catechol-O-methyl transferase activity in aorta were determined 18 hours, 4 days or 7 days later. Norepinephrine uptake and enzyme activities in the aorta were not altered by ricin administration. Norepinephrine release and content of aorta were increased at most time periods following ricin administration, significantly so for NE content at 4 days and for release at 18 hours following the lower dose of ricin. We conclude that the mechanisms involved in the release of NE from sympathetic nerves in the vasculature are not impaired by ricin administration, but rather show changes that indicate increased compensatory activity.

The cardiovascular effects of ricin in rabbits.

Ricin is a toxic lectin from the castor bean. The time course of its toxic effects on the cardiovascular system of rabbits was investigated after determining its LD50 and minimum lethal dose in rabbits by the Up and Down method, as a basis for dosing. Systolic and diastolic arterial pressures, electrocardiogram and heart rate were recorded for 48 hr following administration of either a toxic sublethal (0.22 microgram/kg) or minimum lethal dose (0.44 microgram/kg) of ricin. After a delayed onset of about 20 hr, the minimum lethal dose (0.44 microgram/kg) of ricin caused a significant decrease in both systolic and diastolic pressures (P < 0.05). The systolic and diastolic pressures decreased 0.47 and 0.39 mmHg/hr more so than control groups, respectively. A toxic sublethal dose (0.22 microgram/kg) of ricin did not significantly alter either systolic or diastolic pressure. Neither dose of ricin caused cardiac arrhythmias or significantly increased heart rate. We conclude that the lethal hypotensive sequelae of ricin toxicity in the rabbit were peripheral in origin and not cardiogenic at these two doses.

Dibenamine enhancement of histamine-induced relaxation of the rabbit mesenteric artery.

Helically cut strips of rabbit mesenteric artery relax when exposed to histamine if their histamine H1 receptors are first blocked by 7 X 10(-6) M mepyramine. Relaxations are potentiated by 20 min pretreatment with 10(-6) M dibenamine. This dibenamine regimen also enhances relaxation of the strips to the selective H2 receptor agonist dimaprit, and to a lesser extent to papaverine which does not act on histamine receptors. This enhancement occurs both at 38 degrees and 22 degrees, and in mesenteric artery strips from rabbits reserpinized to deplete amine stores. Histamine has a greater relaxant effect on mesenteric artery strips at 22 degrees than at 38 degrees, normally. Dibenamine-treated strips do not relax more at the lower temperature, however. Thus, dibenamine nonselectively enhances relaxations of mesenteric artery and may enhance histamine-induced relaxations by an additional mechanism.

Rabbit mesenteric artery receptors formed by cold exposure mediating increased histamine-induced relaxation.

On helically-cut strips of rabbit mesenteric artery contracted with phenylephrine, histamine in the presence of mepyramine caused a partial relaxation at 42 degrees C which was potentiated by reducing the temperature to 25 degrees C. However, under the same conditions, the selective H2 agonist, dimaprit, caused a smaller partial relaxation not enhanced by cooling the strips. Neither changing the temperature range (to 38 degrees and 22 degrees C), deleting the H1 blocker, nor substitution of another H1 blocker (pyribenzamine) resulted in an enhanced relation to dimaprit in the cold. Prior reserpinization of the rabbit did not abolish the cooling-enhanced relaxation to histamine, but did abolish the reversal of relaxation observed at high (10(-3) M) histamine concentrations. Metiamide (3 X 10(-4) M) blocked both histamine (H2)- and dimaprit-induced relaxations more effectively in cooled strips. Thus the H2 receptor activity observed in cooled strips differs in selectivity for the two agonists and antagonist from that observed at normal temperature.