Binding site of α2-plasmin inhibitor to plasminogen

Alpha-2-antiplasmin (␣2AP) undergoes both N-and C-terminal cleavages, which significantly modify its activities. Compared with other Ser protease inhibitors (serpins), ␣2AP contains an ϳ 50-residueextended C-terminus, which binds plasmin(ogen). We developed 2 new ELISAs to measure the antigen levels of free total ␣2AP and free C-terminally intact ␣2AP to investigate whether ␣2AP antigen levels or ␣2AP C-terminal cleavage were associated with myocardial infarction (MI) in 320 male MI survivors and 169 age-

Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Proteolytic and genetic variation of the alpha-2-antiplasmin C-terminus in myocardial infarction

Willige

Miedzak

Carter

et al. 2011

“…It should be noted that normal human plasma that had been depleted of ␣ 2 AP using immobilized polyclonal antibodies to ␣ 2 AP was used in these studies, thereby negating any competitive impact that C-terminal shortened, nonplasminogen-binding forms of ␣ 2 AP may exhibit for becoming cross-linked to fibrin. 18,19 The Met-␣ 2 AP and Asn-␣ 2 AP added to ␣ 2 AP-depleted plasma were purified using their C-terminal affinities for immobilized plasminogen kringles 1-3; hence, C-terminal degraded forms of ␣ 2 AP would not bind. These studies demonstrate clearly that Asn-␣ 2 AP inhibits plasmin-mediated fibrin digestion more effectively than Met-␣ 2 AP.…”

Section: Effect Of Met-␣ 2 Ap or Asn-␣ 2 Ap On Plasma Clot Lysismentioning

confidence: 99%

A novel plasma proteinase potentiates α2-antiplasmin inhibition of fibrin digestion

Lee

Jackson

Christiansen

et al. 2004

118

164

Human ␣ 2 -antiplasmin (␣ 2 AP), also known as ␣ 2 -plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. There are 2 N-terminal forms of ␣ 2 AP that circulate in human plasma: a 464-residue protein with Met as the N-terminus, Met-␣ 2 AP, and a 452-residue version with Asn as the N-terminus, Asn-␣ 2 AP. We have discovered and purified a proteinase from human plasma that cleaves the Pro12-Asn13 bond of Met-␣ 2 AP to yield Asn-␣ 2 AP and have named it antiplasmin-cleaving enzyme (APCE). APCE is similar in primary structure and catalytic properties to membranebound fibroblast activation protein/seprase for which a physiologic substrate has not been clearly defined. We found that Asn-␣ 2 AP becomes cross-linked to fibrin by activated factor XIII approximately 13 times faster than native Met-␣ 2 AP during clot formation and that clot lysis rates are slowed in direct proportion to the ratio of Asn-␣ 2 AP to Met-␣ 2 AP in human plasma. We conclude that APCE cleaves Met-␣ 2 AP to the derivative Asn-␣ 2 AP, which is more efficiently incorporated into fibrin and consequently makes it strikingly resistant to plasmin digestion. APCE may represent a new target for pharmacologic inhibition, since less generation and incorporation of Asn-␣ 2 AP could result in a more rapid removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states. IntroductionHuman ␣ 2 -antiplasmin (␣ 2 AP), also known as ␣ 2 -plasmin inhibitor, is the main inhibitor of plasmin. 1 Plasmin plays a critical role in fibrin proteolysis and tissue remodeling. The physiologic relevance of plasmin inhibition by ␣ 2 AP to blood clotting and fibrinolytic homeostasis is supported by the following observations: (1) the rate of free plasmin inactivation by circulating ␣ 2 AP is much faster than fibrin(ogen) digestion by plasmin, 2 thereby eliminating the possibility of a systemic lytic state and consequent bleeding; (2) ␣ 2 AP is cross-linked to forming fibrin by activated blood clotting factor XIII (FXIIIa) and inhibits plasmin-mediated lysis in direct proportion to the amount incorporated 3-5 ; and (3) patients with homozygous ␣ 2 AP deficiency manifest serious hemorrhagic tendencies, while heterozygotes tend to bleed only after major trauma or surgery. 6 Human ␣ 2 AP is synthesized primarily in the liver, and during circulation in plasma, the secreted precursive Met-␣ 2 AP form, a 464-residue protein having Met as the N-terminus, undergoes proteolytic cleavage between Pro12 and Asn13 to yield Asn-␣ 2 AP, a 452-residue version with Asn as the N-terminus. 7 Met-␣ 2 AP accounts for approximately 30% of circulating ␣ 2 AP, and Asn-␣ 2 AP, approximately 70%. 7,8 While 3-fold more Asn-␣ 2 AP than recombinant Met-␣ 2 AP was shown to cross-link to fibrin, 9 no data have been reported for native circulating Met-␣ 2 AP. Moreover, the effect of different ratios of the 2 ␣ 2 AP forms on clot lysis has not been reported. Finally, the enzyme responsible for converting Met-␣ 2 AP to Asn-␣ 2 AP has not been ident...

“…81 Although some subsequent studies have reported this trypsin cleavage site as the position where PB-a2AP is truncated into NPB-a2AP, to date it is unknown where and how the conversion happens in the circulation. An actual in vivo cleavage site and the protease responsible have not yet been identified, despite previous attempts to inhibit NPB-a2AP formation during plasma incubation by a spectrum of protease inhibitors directed to various serine and thiol proteases.…”

mentioning

confidence: 99%

“…80 A 26-amino-acid peptide cleavage product representing the C-terminal 26 amino acids of the molecule was found in PB-a2AP but was not present in NPB-a2AP formed in vitro, which indicates its participation in plasminogen binding. 81 Although some subsequent studies have reported this trypsin cleavage site as the position where PB-a2AP is truncated into NPB-a2AP, to date it is unknown where and how the conversion happens in the circulation. An actual in vivo cleavage site and the protease responsible have not yet been identified, despite previous attempts to inhibit NPB-a2AP formation during plasma incubation by a spectrum of protease inhibitors directed to various serine and thiol proteases.…”

mentioning

confidence: 99%

Natural heterogeneity of α2-antiplasmin: functional and clinical consequences

Abdul

Leebeek

Rijken

et al. 2016

Human a2-antiplasmin (a2AP, also called a2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. a2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced a2AP levels in hereditary a2AP deficiency may lead to bleeding symptoms, whereas increased a2AP levels have been associated with increased thrombotic risk. a2AP is a very heterogeneous protein. In the circulation, a2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of a2AP is cleaved at the N terminus by antiplasmin-cleaving enzyme (or soluble fibroblast activation protein), resulting in a 12-amino-acid residue shorter form. The glutamine residue that serves as a substrate for activated factor XIII becomes more efficient after removal of the N terminus, leading to faster crosslinking of a2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating a2AP, the C terminus is absent. This C terminus contains the binding site for plasmin(ogen), the key component necessary for the rapid and efficient inhibitory mechanism of a2AP. Without its C terminus, a2AP can no longer bind to the lysine binding sites of plasmin(ogen) and is only a kinetically slow plasmin inhibitor. Thus, proteolytic modifications of the N and C termini of a2AP constitute major regulatory mechanisms for the inhibitory function of the protein and may therefore have clinical consequences. This review presents recent findings regarding the main aspects of the natural heterogeneity of a2AP with particular focus on the functional and possible clinical implications. (Blood. 2016; 127(5):538-545) Introduction a2-antiplasmin (a2AP, also called a2-plasmin inhibitor) is a key player in the fibrinolytic system (Figure 1). The fibrinolytic system is crucial for dissolving fibrin clots, facilitating tissue repair, and preventing clots from occluding vessels.1 Recent clinical studies have shown that reduced fibrinolysis (eg, due to an increase in a2AP level) is associated with an increase in both venous and arterial thrombotic risk. 2In contrast, an increase in fibrinolysis due to a2AP deficiency is associated with hemophilia-like bleeding symptoms, which typically occur after initial hemostasis as a result of the premature dissolution of fibrin. The phenotype of a2AP deficiency is heterogeneous. Complete congenital a2AP deficiency leads to severe bleeding with hemophilialike bleeding symptoms such as joint bleeding, whereas heterozygous a2AP-deficient patients typically have mild or no bleeding symptoms. 3,4 In addition, acquired a2AP deficiency may also occur in liver disease 5 and amyloidosis 6 or during fibrinolytic therapy. 7The main enzyme of the fibrinolytic system is the serine protease plasmin, which is predominantly responsible for the degradation of fibrin into rapidly cleared soluble fibrin degradation products (Figure 1). The inactive proenzyme plasminogen ca...