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...

Objective: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. Methods and results: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4 −/− and CD80 −/− CD86 −/− mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p = 0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p = 0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1 +, PD1 +, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. Conclusions: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.

To cite this article: Rijken DC, Abdul S, Malfliet JJMC, Leebeek FWG, Uitte de Willige S. Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII. J Thromb Haemost 2016; 14: 1453-61. Essentials• Factor XIIIa inhibits fibrinolysis by forming fibrinfibrin and fibrin-inhibitor cross-links.• Conflicting studies about magnitude and mechanisms of inhibition have been reported.• Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots.• Cross-links of a2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot.Summary. Background: Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links a 2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective: To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results: Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of a 2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of a 2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions: Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of a 2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction.

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In young female VWD patients, we observed that low PAI-1 levels were associated with a higher bleeding score, which may partly explain the observed variability in bleeding phenotype in VWD patients.