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In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10 μmol/l) or CO (carbon monoxide-releasing molecule-2, 100 μmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.

Cigarette smoke and carbon monoxide (CO) released from tricarbonyldichlororuthenium (II) dimer (CORM-2) attenuate fibrinolysis. The purpose of the present study was to determine whether CO diminished fibrinolysis by enhancement of α₂-antiplasmin via a putative heme group. Plasma, isolated α₂-antiplasmin and isolated plasmin were exposed to CO released from CORM-2 and nitric oxide (NO) via a NO donor to induce carboxyheme and metheme states, respectively. Exposed, isolated enzymes were placed in either α₂-antiplasmin-deficient or normal plasma. Effects of CO and NO on tissue-type plasminogen activator initiated fibrinolysis were determined by thrombelastography. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify heme released from α₂-antiplasmin and plasmin. CO significantly enhanced α₂-antiplasmin activity, but decreased plasmin activity. NO decreased both α₂-antiplasmin and plasmin activity. Although inadequate LC-MS/MS data were obtained with α₂-antiplasmin (secondary to glycosylation), a putative plasmin-associated heme was identified. CO elicits hypofibrinolysis by enhancing α₂-antiplasmin activity and decreasing plasmin activity. On the basis of the responses to NO and LC-MS/MS data, it is highly likely that both enzymes are modulated by attached heme groups. Efforts to develop methods to detect CO-mediated hypercoagulability are ongoing, with the goal of identifying populations at risk of thrombotic morbidity secondary to cigarette smoking.

The objective of this study was to determine whether carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer, CORM-2) directly affects α2-antiplasmin activity. For this purpose, purified α2-antiplasmin was exposed to 0 or 100 μmol/l CORM-2 for 5 min at 37°C and then placed in α2-antiplasmin-deficient plasma (25 μg/ml α2-antiplasmin and 3.3 μmol/l CORM-2 final concentrations). In a second series of experiments, α2-antiplasmin and deficient plasma were combined and then exposed to 0 or 100 μmol/l CORM-2. Coagulation was activated with tissue factor and fibrinolysis initiated with tissue-type plasminogen activator (n = 8 per condition). Thrombus growth/disintegration kinetics were monitored with thrombelastography until clot lysis time occurred. Samples containing α2-antiplasmin preexposed to 100 μmol/l CORM-2 demonstrated no changes in the velocity of clot growth, but had a significant prolongation of the time to maximum rate of lysis, clot lysis time, and a significant decrease in the maximum rate of clot lysis compared with samples preexposed to 0 μmol/l CORM-2. In sharp contrast, addition of 100 μmol/l CORM-2 to premixed α2-antiplasmin in its deficient plasma resulted in significant, marked increases in the velocity of clot growth and the strength with concurrent antifibrinolytic effects as in the first series. In conclusion, CORM-2 exerts its antifibrinolytic effects by direct enhancement of α2-antiplasmin activity. It appears that combined modification of both fibrinogen and α2-antiplasmin are responsible for the robust procoagulant/antifibrinolytic effects of CORM-2 in the fibrinolytic environment.

Carbon monoxide, derived from carbon monoxide-releasing molecules, has been recently demonstrated to enhance the velocity of formation and strength of plasma thrombi. We tested the hypothesis that carbon monoxide-releasing molecule-2 would modulate fibrinolysis of plasma thrombi. Normal plasma was exposed to 0, 25, 50, 100 or 200 micromol/l carbon monoxide-releasing molecule-2, with coagulation activated with tissue factor and fibrinolysis initiated with tissue-type plasminogen activator. Additional experiments utilized factor XIII, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor or alpha2-antiplasmin-deficient plasmas. Thrombus growth/disintegration kinetics was monitored with thrombelastography. Carbon monoxide-releasing molecule-2, in a concentration-dependent fashion, increased the velocity of thrombus formation and strength, and markedly attenuated fibrinolysis in normal plasma. In factor XIII-deficient plasma, carbon monoxide-releasing molecule-2 mediated effects on thrombus growth/disintegration kinetics were similar to that seen with normal plasma; however, carbon monoxide-releasing molecule-2 had a less marked effect on thrombus growth/disintegration in both plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor-deficient plasma, with even less carbon monoxide-releasing molecule-2-mediated effects noted in alpha2-antiplasmin-deficient plasma. Carbon monoxide-releasing molecule-2 attenuated fibrinolysis by enhancing the velocity of clot growth and strength while augmenting the effects of plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and alpha2-antiplasmin. These findings serve as the rationale for further investigations to determine if carbon monoxide-releasing molecules could be utilized as hemostatic agents.

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.

Stabilized, active plasmin is a novel thrombolytic for direct delivery to clots. Although it is known that protease inhibitors in plasma inhibit plasmin, the amount of plasmin that can be added to plasma/blood before free plasmin is observed is not clear. Determination of free plasmin activity in plasma using chromogenic substrates represents a challenge due to false-positive signals from plasmin entrapped by alpha2-macroglobulin. Size-exclusion chromatography was used to separate the plasmin-alpha2-macroglobulin complex from uninhibited, free plasmin. In this in-vitro study, exogenous plasmin is effectively inhibited up to 2.4 micromol/l after 5-min incubation with plasma at 37 degrees C. Initially, plasmin was consumed predominantly by alpha2-antiplasmin up to 1.2 micromol/l plasmin. Following exhaustion of alpha2-antiplasmin, plasmin was further consumed by alpha2-macroglobulin up to 2.4 micromol/l plasmin added to human plasma. Whole human blood was found to have an increased inhibitory capacity over that of plasma; free plasmin activity could be measured only above 3.8 micromol/l added plasmin. In conclusion, several mechanisms exist that control plasmin activity in human blood; in addition to alpha2-antiplasmin and alpha2-macroglobulin, blood cells contribute to the inhibition of exogenously administered plasmin. These in-vitro results indicate that doses of plasmin up to approximately 12 mg/kg in humans can be completely inactivated by blood.

Two major proteins that inhibit fibrinolysis include thrombin activatable fibrinolysis inhibitor (TAFI) and alpha2-antiplasmin. Our goal was to quantify the contribution of TAFI and alpha2-antiplasmin to antifibrinolytic defenses with thrombelastography. Plasma activated with tissue factor/kaolin was subjected to fibrinolysis with tissue-type plasminogen activator (100 U/ml). Prior to activation, TAFI activity was inhibited with either potato carboxypeptidase inhibitor (25 microg/ml) or an anti-TAFI antibody, and alpha2-antiplasmin activity was inhibited with an anti-alpha2-antiplasmin antibody. Data were collected for 30 min, with the time of onset and rate of fibrinolysis determined. Compared with uninhibited samples, TAFI inhibition significantly (P < 0.05) decreased the time of onset of fibrinolysis by 70% and increased the rate of lysis by 70%. There was no difference between potato carboxypeptidase inhibitor and anti-TAFI antibody inhibition. Inhibition of alpha2-antiplasmin resulted in a significantly (P < 0.05) decreased time of onset (85%) and increased the rate of lysis (557%) compared with uninhibited samples. Inhibition of alpha2-antiplasmin activity resulted in a significantly (P < 0.05) greater fibrinolytic response than TAFI inhibition. In conclusion, utilization of standard inhibitors and thrombelastography permitted quantification of the effects of TAFI and alpha2-antiplasmin on fibrinolysis in plasma. Future investigation of diseases involving hypofibrinolysis (e.g. left ventricular assist devices) could be conducted using this assay system.

This study investigates, by slow binding kinetics methods, reaction kinetics of both plasmin types 1 and 2 with alpha -antiplasmin in the presence of increasing concentrations of either epsilon-amino-caproic acid (EACA) or soluble fibrin. All curves of plasmin-alpha -antiplasmin interaction followed the same pattern, indicating reversible slow binding inhibition with an initial loose complex and a following tight complex. Without soluble fibrin or EACA, differences between plasmin types 1 and 2 could be seen in the initial loose complex formation. The presence of increasing concentrations of EACA slowed down the first step of the reaction (without any effect on the second step), resulting in increasing values for K. Plasmin type 1 demonstrated a steep slope of K at an EACA concentration of 1 mmol/l. In plasmin type 2, the increase of K started at higher EACA concentrations. The value for K at a high EACA concentration (100 mmol/l) was the same for both plasmin types. In contrast to EACA, increasing concentrations of soluble fibrin slowed down both reaction steps. At high concentrations of soluble fibrin, the inhibitory effect of alpha -antiplasmin was almost completely abolished. Our data demonstrate that the effect of soluble fibrin and the lysine analogue EACA on plasmin-antiplasmin reactions are different and that the use of lysine analogues does not mimic fibrin in laboratory analyses of plasmin inhibition. In addition, our data indicate theoretical differences between plasmin type 1 and plasmin type 2, when used for local thrombolytic therapy.(2) (2) (i initial) (i initial) (i initial) (i initial) (2)

Staphylokinase (SAK), produced by Staphylococcus aureus, induces fibrinolytic activity in circulation without systemic fibrinolytic activation. Since the effect of blood vessels on the activity of SAK has not yet been clarified, plasminogen activator (PA) activity of SAK in the presence or absence of endothelial cells was analyzed. The endothelial cells used in this experiment were of a cloned established cell line (TKM-33). In the expression of PA activity by SAK or streptokinase (SK), the kinetic constants revealed as Vmax/km were increased about 1.5-fold in the presence of endothelial cells. Furthermore, an initial lag phase which was observed during the plasminogen activation by SAK was markedly shortened in the presence of endothelial cells. In the case of SK, an initial lag phase was not observed in the absence or presence of endothelial cells. Although PA activity of SAK was inhibited by alpha 2-antiplasmin (alpha 2-AP), the inhibitory effect of alpha 2-AP in the presence of endothelial cells was weaker than in the absence of endothelial cells. The cyanogen bromide digested fibrinogen fragment-2 (FCB-2) distinctly enhanced the PA activity of SAK in the absence and the presence of endothelial cells. However, alpha 2-AP and FCB-2 did not cause a significant alteration of PA activity of SK even in the absence or presence of endothelial cells. These findings suggest that PA activity of SAK is enhanced by endothelial cells, but inhibited by alpha 2-AP. Moreover, PA activity of SAK is further enhanced by fibrin clot in the presence of endothelial cells.

The aim of this study was to synthesize and investigate hybrid peptides which contain the RGD (Arg-Gly-Asp) sequence coupled with lysine residues in special arrangements (antiplasmin carboxyterminal peptide) in an effort to simultaneously inhibit platelet aggregation and promote fibrinolysis. The in vitro haemostatic modifying properties of the synthesized peptides were tested by ADP-induced platelet aggregation, plasmin-generation tests and fibrin-clot lysis assays. The hybrid peptide RGDFAP, composed of RGDF (Arg-Gly-Asp-Phe) coupled to a synthetic peptide residue of the carboxyterminal part of antiplasmin (AP26) inhibited platelet activation and increased plasmin generation and in vitro fibrin-clot lysis.