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

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3783-3788 ◽  
Author(s):  
Kyung N. Lee ◽  
Kenneth W. Jackson ◽  
Victoria J. Christiansen ◽  
Keun H. Chung ◽  
Patrick A. McKee
Keyword(s):  
Human Plasma ◽  
Catalytic Properties ◽  
Fibroblast Activation Protein ◽  
Clot Lysis ◽  
Direct Proportion ◽  
N Terminus ◽  
Rapid Removal ◽  
Membrane Bound ◽  
Plasmin Inhibitor ◽  
Pharmacologic Inhibition

Abstract Human α2-antiplasmin (α2AP), also known as α2-plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. There are 2 N-terminal forms of α2AP that circulate in human plasma: a 464-residue protein with Met as the N-terminus, Met-α2AP, and a 452-residue version with Asn as the N-terminus, Asn-α2AP. We have discovered and purified a proteinase from human plasma that cleaves the Pro12-Asn13 bond of Met-α2AP to yield Asn-α2AP and have named it antiplasmin-cleaving enzyme (APCE). APCE is similar in primary structure and catalytic properties to membrane-bound fibroblast activation protein/seprase for which a physiologic substrate has not been clearly defined. We found that Asn-α2AP becomes cross-linked to fibrin by activated factor XIII approximately 13 times faster than native Met-α2AP during clot formation and that clot lysis rates are slowed in direct proportion to the ratio of Asn-α2AP to Met-α2AP in human plasma. We conclude that APCE cleaves Met-α2AP to the derivative Asn-α2AP, 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-α2AP could result in a more rapid removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states.

scholarly journals Different N-terminal forms of α2-plasmin inhibitor in human plasma

1993 ◽  
Vol 291 (2) ◽  
pp. 623-625 ◽  
Author(s):  
K Bangert ◽  
A H Johnsen ◽  
U Christensen ◽  
S Thorsen
Keyword(s):  
Human Plasma ◽  
Cdna Sequence ◽  
Recognition Site ◽  
Signal Peptidase ◽  
Stable Complex ◽  
N Terminus ◽  
Functional Implications ◽  
Plasmin Inhibitor

Mature alpha 2-plasmin inhibitor in human plasma has 12 more N-terminal residues than hitherto anticipated. The first residue is the methionine at position 28, downstream from the N-terminus of the pre-protein. The cDNA sequence predicts that the site cleaved upon formation of the mature inhibitor is a typical signal-peptidase recognition site. The mature inhibitor (464 residues) and the previously reported, and presumably degraded, form with N-terminal asparagine (452 residues), are present in plasma in about equal amounts. They both form a stable complex with plasmin. Recent studies on a recombinant alpha 2-plasmin inhibitor suggest that the 12 additional residues have functional implications [Sumi, Ichikawa, Nakamura, Miura and Aoki (1989) J. Biochem. 106, 703-707].

scholarly journals Natural heterogeneity of α2-antiplasmin: functional and clinical consequences

Blood ◽  
2016 ◽  
Vol 127 (5) ◽  
pp. 538-545 ◽  
Author(s):  
Shiraazkhan Abdul ◽  
Frank W. G. Leebeek ◽  
Dingeman C. Rijken ◽  
Shirley Uitte de Willige
Keyword(s):  
Fibrin Clot ◽  
Clot Lysis ◽  
Thrombotic Risk ◽  
Amino Terminal ◽  
Inhibitory Function ◽  
C Terminus ◽  
N Terminus ◽  
Clinical Consequences ◽  
Carboxyl Terminal ◽  
Plasmin Inhibitor

AbstractHuman α2-antiplasmin (α2AP, also called α2-plasmin inhibitor) is the main physiological inhibitor of the fibrinolytic enzyme plasmin. α2AP inhibits plasmin on the fibrin clot or in the circulation by forming plasmin-antiplasmin complexes. Severely reduced α2AP levels in hereditary α2AP deficiency may lead to bleeding symptoms, whereas increased α2AP levels have been associated with increased thrombotic risk. α2AP is a very heterogeneous protein. In the circulation, α2AP undergoes both amino terminal (N-terminal) and carboxyl terminal (C-terminal) proteolytic modifications that significantly modify its activities. About 70% of α2AP 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 α2AP to fibrin and consequently prolonged clot lysis. In approximately 35% of circulating α2AP, 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 α2AP. Without its C terminus, α2AP 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 α2AP 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 α2AP with particular focus on the functional and possible clinical implications.

Different mechanisms contribute to the biphasic pattern of carboxypeptidase U (TAFIa) generation during in vitro clot lysis in human plasma

2003 ◽  
Vol 89 (02) ◽  
pp. 264-271 ◽  
Author(s):  
Judith Leurs ◽  
Britt-Marie Wissing ◽  
Viveca Nerme ◽  
Katinka Schatteman ◽  
Petter Björquist ◽  
...  
Keyword(s):  
Human Plasma ◽  
Time Course ◽  
Thrombin Inhibitor ◽  
Clot Lysis ◽  
Time Period ◽  
Lysis Time ◽  
Two Peaks ◽  
Plasmin Inhibitor ◽  
Clot Lysis Time

SummaryCarboxypeptidase U (CPU, TAFIa) recently gained interest as a significant player in dampening the fibrinolytic rate. The aim of this study was to investigate the time course of the generation of CPU activity during coagulation and fibrinolysis using an in vitro clot lysis model in human plasma. A first peak of CPU activity appeared after initiation of the coagulation phase and a second rise in CPU activity was observed during the fibrinolysis. The decrease in the proCPU plasma concentration followed the same trend as the appearance of the CPU activity. The direct thrombin inhibitor inogatran eliminated the CPU generation during coagulation but not during fibrinolysis. Addition of the plasmin inhibitor aprotinin during fibrinolysis resulted in a decrease in CPU activation during the lysis phase. These results demonstrate that proCPU was activated during coagulation by thrombin and during fibrinolysis by plasmin. Addition of a CPU inhibitor before initiation of clotting decreased the clot lysis time as expected. However, addition in the time period between the two peaks of CPU activity had no apparent effect on the clot lysis time.

scholarly journals Circulating FGF21 proteolytic processing mediated by fibroblast activation protein

2016 ◽  
Vol 473 (5) ◽  
pp. 605-614 ◽  
Author(s):  
Eugene Y. Zhen ◽  
Zhaoyan Jin ◽  
Bradley L. Ackermann ◽  
Melissa K. Thomas ◽  
Jesus A. Gutierrez
Keyword(s):  
Human Plasma ◽  
Proteolytic Cleavage ◽  
Fibroblast Activation Protein ◽  
Proteolytic Processing ◽  
Fibroblast Activation ◽  
Dpp Iv ◽  
N Terminus

Proteolytic cleavage of FGF21, evaluated in human plasma, confirmed three known proline hydrolysis sites. Although DPP-IV participates in consecutive cleavages from the N-terminus, fibroblast activation protein was implicated as the enzyme responsible for the C-terminal cleavage that inactivates FGF21.

Functional Properties of p-Anisoylated Plasmin-Staphylokinase Complex

1993 ◽  
Vol 70 (02) ◽  
pp. 326-331 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
R A G Smith ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Rate Constant ◽  
Time Course ◽  
Bolus Injection ◽  
Similar Rate ◽  
Clot Lysis ◽  
Lag Phase ◽  
Plasma Clot ◽  
Rapid Clearance ◽  
Stoichiometric Complex

SummaryThe kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p’-anisate-HCI was 52 M-1 s-1 and its deacylation rate constant 1.2 × 10-4 s-1 (t½ of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min.The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.

Inhibition of Coagulation and Fibrinolysis by Aromatic Amidino Compounds

1971 ◽  
Vol 25 (03) ◽  
pp. 391-404 ◽  
Author(s):  
J.D Geratz
Keyword(s):  
Prothrombin Time ◽  
Human Plasma ◽  
Partial Thromboplastin Time ◽  
Inhibitory Effect ◽  
Clot Lysis ◽  
Similar Degree ◽  
Contact Activation ◽  
Plasma Clot ◽  
Human Thrombin ◽  
Canine Plasma

Summary1. Aromatic diamidines which are potent inhibitors of trypsin possess a marked inhibitory effect on the clotting activity of human thrombin and on the prothrombin time and partial thromboplastin time of human plasma. They also block the contact activation phase of the coagulation process. The strongest inhibitor among the compounds tested was M & B 4596 which was followed in second place by pentamidine.2. Pentamidine was 10 times more active than ε-ACA in impeding streptokinase-induced lysis of human plasma clots. It was 100-200 times stronger than ε-ACA in inhibiting the activation of bovine plasminogen by activators formed from the interaction between streptokinase and either human plasmin(ogen) or human plasma.3. The prothrombin time and partial thromboplastin time of canine plasma were less susceptible to inhibition by pentamidine than the same tests on human plasma. Clot lysis in the canine system was inhibited by pentamidine to a similar degree as in the human system. After intravenous injection of pentamidine in the dog there occurred the expected prolongation of the partial thromboplastin time and of the clot lysis time.

Inhibition of the Activities of α2-Plasmin inhibitor (PI) and CI inhibitor (CI-Inh) by the Synthetic Fibrinolytic Agent, 3-Hydroxypropyl Flufenamide (Flu-HPA)

1979 ◽  
Author(s):  
L Miles ◽  
J Burnier ◽  
M Verlander ◽  
M Goodman ◽  
A Kleiss ◽  
...  
Keyword(s):  
Plasminogen Activators ◽  
Inhibitory Effect ◽  
Mechanisms Of Action ◽  
Flufenamic Acid ◽  
Clot Lysis ◽  
Factor Xiia ◽  
Dependent Inhibition ◽  
Normal Human ◽  
Plasmin Inhibitor

Flu-HPA is one of a series of flufenamic acid derivations that enhances plasminogen-dependent clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The influence of Flu-HPA on the inhibition of purified plasmin by purified PI was studied. PI activity was assessed by its inhibition of the clevage of the tripeptide S-2251 (H-D-Val-Leu-Lys-pNA) by plasmin. Flu-HPA was dissolved in DMF or in methonol and preincubated with PI before addition of plasmin. At Flu-HPA concentrations greater than 1mM and up to 60mM, the inhibitory activity of PI was totally lost. The inhibitory effect of normal human plasma on plasmin was also completely abolished at concentrations of Flu-HPA between 2.5 and 40mM. The effect of Flu-HPA on the inhibition of purified plasma kallikrein by purified CI-Inh was also studied. CI-Inh activity was measured by its inhibition of cleavage of the tripeptide Bz-Pro-Phe-Arg-pNA by kallikrein. When Flu-HPA, dissolved in DMF or in methonol, was preincubated with CI-Inh, a concentration dependent inhibition of CI-Inh activity was observed. CI-Inh activity was abolished by concentrations of Flu-HPA greater than 1mM. Flu-HPA also inhibited the activity of CI-Inh on purified Factor XIIa. These observations suggest that this flufenamic acid derivative may enhance fibrinolysis not only by inhibiting PI activity but also by decreasing the inactivation of plasminogen activators by CI-Inh.

Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

1986 ◽  
Vol 56 (01) ◽  
pp. 035-039 ◽  
Author(s):  
D Collen ◽  
F De Cock ◽  
E Demarsin ◽  
H R Lijnen ◽  
D C Stump
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Dose Response ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

scholarly journals Incorporation of α2-Plasmin Inhibitor into Fibrin Clots and Its Association with the Clinical Outcome of Acute Ischemic Stroke Patients

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 347
Author(s):  
Zsuzsa Bagoly ◽  
Barbara Baráth ◽  
Rita Orbán-Kálmándi ◽  
István Szegedi ◽  
Réka Bogáti ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Acute Ischemic Stroke ◽  
Intracranial Hemorrhage ◽  
Intravenous Thrombolysis ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Stroke Patients ◽  
Plasmin Inhibitor ◽  
Fibrin Clots

Cross-linking of α2-plasmin inhibitor (α2-PI) to fibrin by activated factor XIII (FXIIIa) is essential for the inhibition of fibrinolysis. Little is known about the factors modifying α2-PI incorporation into the fibrin clot and whether the extent of incorporation has clinical consequences. Herein we calculated the extent of α2-PI incorporation by measuring α2-PI antigen levels from plasma and serum obtained after clotting the plasma by thrombin and Ca2+. The modifying effect of FXIII was studied by spiking of FXIII-A-deficient plasma with purified plasma FXIII. Fibrinogen, FXIII, α2-PI incorporation, in vitro clot-lysis, soluble fibroblast activation protein and α2-PI p.Arg6Trp polymorphism were measured from samples of 57 acute ischemic stroke patients obtained before thrombolysis and of 26 healthy controls. Increasing FXIII levels even at levels above the upper limit of normal increased α2-PI incorporation into the fibrin clot. α2-PI incorporation of controls and patients with good outcomes did not differ significantly (49.4 ± 4.6% vs. 47.4 ± 6.7%, p = 1.000), however it was significantly lower in patients suffering post-lysis intracranial hemorrhage (37.3 ± 14.0%, p = 0.004). In conclusion, increased FXIII levels resulted in elevated incorporation of α2-PI into fibrin clots. In stroke patients undergoing intravenous thrombolysis treatment, α2-PI incorporation shows an association with the outcome of therapy, particularly with thrombolysis-associated intracranial hemorrhage.

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