The regulation of liver regeneration by the plasmin/alpha 2-antiplasmin system

BACKGROUND/AIMS: The regeneration after liver injury is regulated by the release and activation of several growth factors. The role of the plasmin/alpha(2)-antiplasmin (alpha(2)-AP) system in liver regeneration was investigated. METHODS: CCl(4) was injected intraperitoneally into the mice deficient (-/-) in fibrinolytic factors: alpha(2)-AP-/-, plasminogen (Plg) -/-, and Plg-/-.alpha(2)-AP-/-, and wild-type (WT) mice. The liver tissue was examined for its microscopic appearance, fibrinolytic activity, and fibronectin levels. RESULTS: In the gene deficient and WT mice, the livers exhibited the same extent of necrosis 2 days after the CCl(4) injection. The livers of the WT mice normalized after 7 days, and the alpha(2)-AP-/- mice normalized after 5 days. In contrast, the livers of the Plg-/- and Plg-/-.alpha(2)-AP-/- mice remained in the damaged state until 14 days after the liver injury. The injection of anti-alpha(2)-AP antibody in the WT mice improved the regeneration after the liver injury, and the injection of tranexamic acid in the alpha(2)-AP-/- mice reduced. CONCLUSIONS: These results suggest that the plasmin/alpha(2)-AP system played an important role in hepatic repair via clearance from the injury area.     

Lack of alpha 2-antiplasmin promotes re-endothelialization via over-release of VEGF after vascular injury in mice

We here report that the arterial blood flow after endothelial injury in mice deficient in alpha 2-antiplasmin (alpha 2-AP-/- mice) was well maintained compared with that of wild-type mice. Moreover, the development of neointima 4 weeks after injury in alpha 2-AP-/- mice was significantly decreased. Histologic observations showed a prompt recovery of endothelial cells with a much higher proliferating index in repaired endothelium in alpha 2-AP-/- mice. The amount of secreted vascular endothelial growth factor (VEGF) by explanted vascular smooth muscle cells (SMCs) from alpha 2-AP-/- mice was significantly increased. In separate experiments using a human endothelial cell (EC) line, we could demonstrate that plasminogen binds to ECs and that this binding can be prevented by alpha 2-AP. Finally, an injection of either an anti-VEGF receptor-1 antibody or alpha 2-AP reduced the prompt endothelial healing. alpha 2-AP is the main inactivator of plasmin, which cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment. Lack of alpha 2-AP, therefore, could lead to a local over-release of VEGF by the continuously active plasmin in the injured area, which could result in a prompt re-endothelialization after vascular injury. Our results provide new insight into the role of alpha 2-AP and VEGF in the pathogenesis of re-endothelialization following vascular injury.     

Regulation of streptokinase-human plasmin complex by the plasma proteinase inhibitors alpha 2-antiplasmin and alpha 2-macroglobulin is species specific and temperature dependent

Streptokinase-plasmin complex (SkPl) was prepared with human plasminogen. Regulation of SkPl and plasmin by the plasma proteinase inhibitors, alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M), was studied as a function of temperature in plasminogen- depleted human plasma, mouse plasma, and solutions of purified proteins. The reaction of plasmin with proteinase inhibitors in human plasma was complete. alpha 2AP was the predominant inhibitor. The fraction of alpha 2M-plasmin recovered was not affected significantly by incubation temperature. In contrast, the reaction of SkPl with human proteinase inhibitors was markedly temperature dependent. The apparent second-order rate constant for the reaction of SkPl with purified alpha 2AP at 37 degrees C (1.5 x 10(2) mol/L-1 s-1) was greater than 150-fold higher than the constant derived at 4 degrees C. In human plasma and in solutions containing mixtures of purified human proteins, alpha 2AP was the principal inhibitor of SkPl. Elevating the temperature enhanced the reaction of SkPl with alpha 2AP and alpha 2M comparably. Equivalent results were obtained when incubations were performed in platelet-rich plasma (PRP) or whole blood. In murine plasma, SkPl reacted readily with the proteinase inhibitors. The principal inhibitor of SkPl was alpha 2M. Maximum reaction between SkPl and murine alpha 2M was observed at 37 degrees C; however, significant reaction also occurred at 4 degrees C. alpha 2 AP was the predominant inhibitor of plasmin in mouse plasma. Reaction of alpha 2AP with SkPl in murine plasma was significant only after the alpha 2M was inactivated with methylamine. These results were not affected by platelets or whole blood cells. We conclude that the thrombolytic efficacy of streptokinase reflects not only the nature of the plasminogen activator complex but also the function of the proteinase inhibitors.     

Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen

Homozygous plasminogen-deficient (Plg-/-) mice had a significantly reduced thrombolytic capacity toward intravenously injected 125I-fibrin labeled plasma clots prepared from Plg-/- murine plasma (9% +/- 3% lysis after 8 hours; (mean +/- SEM, n = 6), as compared with 82% +/- 8% in wild-type mice; P < .0001). Bolus injection of 1 mg purified murine plasminogen in 10- to 17-week-old Plg-/- mice increased the plasminogen antigen and activity levels at 8 hours to normal levels (130 +/- 5 micrograms/mL). Plasminogen administration was associated with significant restoration of thrombolytic potential (64% +/- 7% spontaneous clot lysis; P < .0001 versus lysis without plasminogen injection). Bolus injection of 1 mg plasminogen in homozygous tissue- type plasminogen activator-deficient (t-PA-/-) mice doubled the plasminogen antigen and activity levels after 8 hours and increased 125I-fibrin clot lysis at 8 hours from 13% +/- 3% to 34% +/- 5% (P = .008). Fibrinogen, t-PA antigen and alpha 2-antiplasmin activity levels after 8 hours were not significantly different in the groups with or without plasminogen injection. Injection of plasminogen induced a variable increase (on average 7- to 10-fold) of PAI-1, but no correlation with the extent of spontaneous clot lysis was observed. Histopathologic examination at the end of the experiments revealed that fibrin deposition in the liver of Plg-/- mice was slightly reduced 8 hours after bolus plasminogen injection (P = .007) and markedly reduced after 24 hours (P < .0001). Plasminogen antigen levels in liver extracts were comparable with those found in wild-type mice at 8 hours (130 +/- 20 versus 110 +/- 15 ng/mg protein) and decreased to 25 +/- 3.2 ng/mg protein at 24 hours. Thus, restoration of normal plasminogen levels in Plg-/- mice normalized the thrombolytic potential toward experimentally induced pulmonary emboli, and resulted in removal of endogenous fibrin deposits within 24 hours.     

Measurements of plasmin-alpha 2 plasmin inhibitor complex and FDP.D dimer levels in the fibrinolytic therapy of acute pulmonary thromboembolism]

The key enzyme for fibrinolysis is plasmin, which is converted from plasminogen by plasminogen activator. Activated plasmin lyses fibrinogen and fibrin to make fibrin degradation products(FDPs) and plasmin is inactivated immediately by alpha 2 plasmin inhibitor. As FDP.D dimer is derived solely from insoluble fibrin, FDP.D dimer is thought of as an index for clot lysis. We measured plasmin-alpha 2 plasmin inhibitor complex(PIC) and FDP.D dimer plasma levels in 3 patients with acute pulmonary thromboembolism treated with recombinant tissue plasminogen activator(tPA). Fifteen million units of tPA(TD-2061) were infused in one hour on the first, second and third hospital days. PIC and FDP.D dimer before tPA infusion showed slightly elevated values as compared to normal ranges. They increased markedly after tPA infusion. These findings suggest that the fibrinolytic system is slightly activated in the acute phase of pulmonary thromboembolism and also strongly activated by tPA infusion. Increased FDP D dimer suggests that fibrin clots are dissolved by activated plasmin. Improvement of arterial oxygen tension was observed after tPA infusion. As sustained higher FDP.D dimer means the existence of fibrin clots, heparin treatment should be continued for prevention of clot formation as long as FDP.D dimer shows higher value. In conclusion, PIC and FDP.D dimer are useful indices not only to detect the activated state of the fibrinolytic system but also to know clot lysis in tPA treatment.     

Mini-plasminogen: a mechanism for leukocyte modulation of plasminogen activation by urokinase

Urokinase activation of blood fibrinolysis involves polymorphonuclear leukocytes. To determine if a leukocyte proteinase can modulate plasminogen activation, plasminogen was digested with leukocyte elastase. A major product was a small, approximately 34,000 dalton fragment (mini-plasminogen), without lysine-binding function, but with fibrin-binding activity. After urokinase activation, the resulting mini- plasmin had amidolytic activity for a tripeptide plasmin substrate and fibrinolytic activity. By 125I-fibrin assay, activities of mini-plasmin and plasmin (12 nmole/liter) were 38 and 20 ng fibrin lysed/min, respectively. Lysis times of fibrin clots containing urokinase, and mini-plasminogen or plasminogen (800 nmole/liter), were 282 and 290 sec, respectively. Mini-plasmin and plasmin were inhibited similarly by epsilon-aminocaproic acid and normal plasma, but differed in responses to gel filtration fractions of plasma containing alpha 2-antiplasmin and alpha 2-macroglobulin, the primary and secondary plasmin inhibitors. With purified inhibitors, mini-plasmin required higher concentrations of, or longer preincubation with, alpha 2-antiplasmin, and lower concentrations of, or shorter preincubation with, alpha 2- macroglobulin, to produce inhibition equivalent to that observed with plasmin. Leukocyte elastase digests plasminogen to generate a mini- plasminogen which, when activated by urokinase, has a novel pattern of response to the major plasmin inhibitors in plasma.     

The fibrinolytic system in man

The fibrinolytic system comprises a proenzyme, plasminogen, which can be activated to the active enzyme plasmin, that will degrade fibrin by different types of plasminogen activators. Inhibition of fibrinolysis may occur at the level of plasmin or at the level of the activators. Fibrinolysis in human blood seems to be regulated by specific molecular interactions between these components. In plasma, normally no systemic plasminogen activation occurs. When fibrin is formed, small amounts of plasminogen activator and plasminogen adsorb to the fibrin, and plasmin is generated in situ. The formed plasmin, which remains transiently complexed to fibrin, is only slowly inactivated by alpha 2-antiplasmin, while plasmin, which is released from digested fibrin, is rapidly and irreversibly neutralized. The fibrinolytic process, thus, seems to be triggered by and confined to fibrin. Thrombus formation may occur as the result of insufficient activation of the fibrinolytic system and (or) the presence of excess inhibitors, while excessive activation and/or deficiency of inhibitors might cause excessive plasmin formation and a bleeding tendency. Evidence obtained in animal models suggests that tissue-type plasminogen activator, obtained by recombinant DNA technology, may constitute a specific clot-selective thrombolytic agent with higher specific activity and fewer side effects than those currently in use.     

Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha2-antiplasmin

This study used two mutants of tissue-type plasminogen activator (t-PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)-1 and alpha2-antiplasmin (alpha2-AP) to the control of fibrin lysis. Wild-type t-PA was compared with KHRR296-299AAAA, which is resistant to PAI-1, and with A473S, which is resistant to alpha2-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha2-AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild-type t-PA or KHRR296-299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of alpha2-AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild-type t-PA; the rate and extent of lysis were enhanced by the addition of antibodies to alpha2-AP or PAI-1. A473S was more effective than wild-type t-PA without the addition of antibodies by virtue of its resistance to alpha2-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha2-AP, even after extended incubation, when 50% of wild-type t-PA could be converted to complex. Comparison of A473S and KHRR296-299AAAA mutants showed their similar effectiveness in lysis of platelet-rich model thrombi. Thus, PAI-1 and alpha2-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha2-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha2-AP as a regulator of fibrin persistence and thrombus resistance to lysis.     

Differential binding of plasminogen to crosslinked and noncrosslinked fibrins: its significance in hemostatic defect in factor XIII deficiency

In spontaneous fibrinolysis of an alpha 2-plasmin inhibitor-deficient plasma clot or tissue-type plasminogen activator-induced fibrinolysis in a purified system without alpha 2-plasmin inhibitor, the lysis was faster when factor XIII-mediated crosslinking of fibrin to fibrin did not occur. During the initial period, the binding of plasminogen to fibrin steadily increased with incubation time. The initial level and subsequent increase of the binding, which may be critical for the subsequent development of fibrinolysis, were more remarkable when fibrin was not crosslinked. The amount of glu- or lys-plasminogen bound to noncrosslinked fibrin was around 4 or 1.5 times larger than the amount of the respective plasminogen bound to crosslinked fibrin. Plasmin was also found to be bound to noncrosslinked fibrin twice as much as the amount bound to crosslinked fibrin. Structural changes induced by crosslinking of fibrin alpha-chain may reduce either the affinity or the number of available complementary sites to lysine binding sites of plasmin(ogen), thereby decreasing the binding of plasmin(ogen) to fibrin. These results suggest that an increased affinity of noncrosslinked fibrin for plasmin(ogen) is contributory to the accelerated fibrinolysis observed in factor XIII deficiency, in addition to an absence of crosslinking of alpha 2-plasmin inhibitor to fibrin.     

A novel plasma proteinase potentiates alpha2-antiplasmin inhibition of fibrin digestion

Human alpha2-antiplasmin (alpha2AP), also known as alpha2-plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. There are 2 N-terminal forms of alpha2AP that circulate in human plasma: a 464-residue protein with Met as the N-terminus, Met-alpha2AP, and a 452-residue version with Asn as the N-terminus, Asn-alpha2AP. We have discovered and purified a proteinase from human plasma that cleaves the Pro12-Asn13 bond of Met-alpha2AP to yield Asn-alpha2AP 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-alpha2AP becomes cross-linked to fibrin by activated factor XIII approximately 13 times faster than native Met-alpha2AP during clot formation and that clot lysis rates are slowed in direct proportion to the ratio of Asn-alpha2AP to Met-alpha2AP in human plasma. We conclude that APCE cleaves Met-alpha2AP to the derivative Asn-alpha2AP, 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-alpha2AP could result in a more rapid removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states.     

The influence of age on in vitro plasmin generation in the presence of fibrin monomer

BACKGROUND AND OBJECTIVES: The components of the fibrinolytic system interact to generate plasmin from its zymogen form, plasminogen. At birth, all the components of the fibrinolytic system are present but with differing plasma concentrations. The present study was undertaken to explore the effect of physiological, age-dependent factors of the fibrinolytic system during childhood on the capacity to generate plasmin. DESIGN AND METHODS: Total plasmin generation was measured in venous plasma from umbilical cords and adults, on plastic and cell surfaces, in the presence of fibrin monomer, Desafib. Plasminogen, its inhibitors alpha2-antiplasmin and plasminogen activator inhibitor type 1, and plasmin-alpha2-antiplasmin complex in the time samples were assayed by enzyme-linked immunosorbent assay. The effect of addition of plasminogen on the plasmin generation in cord plasma and the effect of lipoprotein on adult and cord plasmin generation were measured. RESULTS: On the surface of human umbilical vein endothelial cells, onset of plasmin generation was earlier (40 min) compared to plastic (60 min) but total plasmin generation was similar on both surfaces. The addition of plasminogen to cord plasma increased plasmin generation. Supplementation of lipoprotein in adult plasma had an inhibitory effect, but there was no significant effect in cord plasma. INTERPRETATIONS AND CONCLUSIONS: Plasmin generation is reduced in newborn compared to adult plasma. Decreased plasmin generation in cord plasma is likely due to decreased plasminogen concentration. The antifibrinolytic effect of lipoprotein is more pronounced in adults as compared to newborns due to the presence of higher plasminogen concentration.     

Clot lysis induced by a monoclonal antibody against alpha 2-plasmin inhibitor

A monoclonal antibody (MoAb) to alpha 2-plasmin inhibitor designated JTPI-1 inhibited antiplasmin activity by interfering with formation of alpha 2-plasmin inhibitor (alpha 2-PI)-plasmin complex. With this MoAb, we observed plasma clot lysis in vitro and evaluated the potential of JTPI-1 to serve as a new therapeutic agent for thrombolysis. After adding 125I-labeled fibrinogen to plasma, clots were made by adding thrombin and calcium and were then resuspended in normal plasma containing various concentrations of JTPI-1. The presence of JTPI-1 enhanced release of the soluble 125I-labeled fibrin degradation fragment from the clots in a dose-dependent manner. With tissue plasminogen activator (t-PA)-depleted plasma, we showed that induction of clot lysis by JTPI-1 was dependent on fibrin-bound endogenous t-PA. Regulation of fibrinolysis initiated on the fibrin surface by fibrin-bound t-PA and plasminogen is mediated by alpha 2-PI cross-linked to fibrin by activated factor XIII. JTPI-1 bound to this cross-linked alpha 2-PI neutralized its activity and induced partial digestion of fibrin by plasmin. This resulted in additional binding of Glu-plasminogen to fibrin during the incubation. When 1.2 mumol/L JTPI-1 and 5 U/mL exogenous t-PA were present in the suspending plasma, the rate of clot lysis was essentially the same as that induced by 60 U/mL exogenous t-PA alone. These results suggest that JTPI-1 may be useful in reducing the amount of t-PA administered for thrombolytic therapy.     

Plasmin generation and fibrin(ogen)olysis following desmopressin infusion.

Desmopressin acetate (DDAVP) is known to stimulate the release of tissue-type plasminogen activator (t-PA) from endothelial cells, but it is unclear whether the increased t-PA actually elicits the plasmin generation and fibrin(ogen)olysis in the circulating blood. We measured plasma levels of plasmin-alpha 2-plasmin inhibitor complex, fibrinogen degradation products (FgDP) and fibrin degradation products (FbDP) following desmopressin infusion in 19 patients with bleeding disorders or thrombophilia. Administration of desmopressin (0.3-0.4 microgram/kg) produced a 4.0-fold increase in plasmin-alpha 2-plasmin inhibitor complex at 30 min, whereas neither FgDP nor FbDP was elevated significantly. These findings indicate that desmopressin infusion provokes the generation of plasmin in vivo, but most of the plasmin generated is complexed to alpha 2-plasmin inhibitor and does not degradate fibrin or fibrinogen.     

Regulation by alpha 2-antiplasmin and fibrin of the activation of plasminogen with recombinant staphylokinase in plasma

The effects of alpha 2-antiplasmin and fibrin on the activation of plasminogen by recombinant staphylokinase (STAR) were studied in an effort to elucidate further the molecular basis of the fibrin- specificity of this fibrinolytic agent. In purified systems consisting of 1.5 mumol/L intact or low-M(r) plasminogen and 3 mumol/L alpha 2- antiplasmin, at 37 degrees C and in the absence of fibrin, STAR did not induce plasminogen activation and plasmin-alpha 2-antiplasmin complex (PAP) formation. Addition of a purified fibrin clot (30% vol at a concentration of 3 mg/mL) to mixtures containing intact plasminogen caused approximately 40% plasminogen activation within 2 hours, whereas in mixtures containing low-M(r) plasminogen, no activation was observed. In contrast, 10 nmol/L streptokinase (SK) induced 74% to 100% plasminogen activation within 2 hours in mixtures containing either intact or low-M(r) plasminogen, in both the absence and the presence of fibrin. In citrated human plasma in the absence of fibrin, 30 nmol/L STAR did not induce measurable plasminogen activation and PAP formation (< 1.5% within 2 hours), whereas addition of a plasma clot (12% vol) resulted in complete clot lysis and conversion of 19% +/- 8% of the plasminogen to PAP within 2 hours. Addition of a second plasma clot produced 23% +/- 2% additional plasminogen activation. Equipotent concentrations for plasma clot lysis of SK (100 nmol/L) induced 54% +/- 11% plasminogen activation in the absence and 49% +/- 16% in the presence of fibrin. Addition of 50 mmol/L 6-aminohexanoic acid (6-AHA) abolished the effect of fibrin on plasminogen activation with STAR, but not on activation with SK. In alpha 2-antiplasmin-depleted human plasma in the absence of fibrin, 30 nmol/L STAR did not induce fibrinogen breakdown (> 90% residual fibrinogen after 6 hours), whereas 30 nmol/L preformed plasmin-STAR complex induced extensive fibrinogen degradation (70% within 20 minutes). Thus, in the absence of fibrin, alpha 2- antiplasmin inhibits the activation of plasminogen by STAR, by preventing generation of active plasmin-STAR complex. Fibrin stimulates plasminogen activation by STAR via mechanisms involving the lysine- binding sites of plasminogen, probably by facilitating the generation of plasmin-STAR complex and by delaying its inhibition at the clot surface.     

Alpha2-antiplasmin: potential therapeutic roles in fibrin survival and removal

Alpha2-antiplasmin (alpha2AP) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Two forms of alpha2AP circulate in human plasma: a 464-residue protein with methionine as the amino-terminus (Met-alpha2AP) and an N-terminally-shortened 452-residue form with asparagine as the amino-terminus (Asn-alpha2AP). Human plasma alpha2AP concentration is 1 micro M and consists of approximately 30% Met-alpha2AP and approximately 70% Asn-alpha2AP. The major form (Asn-alpha2AP) is rapidly crosslinked to fibrin during blood clotting by activated coagulation factor XIII and as a consequence, fibrin becomes more resistant to fibrinolysis. It is apparent that alpha2AP is important in modulating the effectiveness and persistence of fibrin with respect to its susceptibility to digestion and removal by plasmin. Hence, the physiologic role of alpha2AP suggests that it may be a useful target for developing more effective treatment of thrombotic diseases. Research on alpha2AP appears to be moving in two main directions: (1) efforts to use variant forms of alpha2AP to reduce bleeding secondary to thrombolytic therapy while not slowing thrombolysis; and (2) efforts to use variant forms to diminish the activity of alpha2AP as a plasmin inhibitor so that fibrinolysis becomes enhanced. Methods to accomplish these two goals mostly involve manipulation of defined functional domains within the molecular structure of alpha2AP, or inhibition of a newly described novel plasma proteinase, termed antiplasmin-cleaving enzyme, that generates the more favorable form of alpha2AP, Asn-alpha2AP, for crosslinking to fibrin. The antiplasmin-cleaving enzyme has similarity in primary structure and catalytic properties to fibroblast activation protein/seprase. This review summarizes recent studies that may hold promise for modulating alpha2AP activity and its interactions with certain proteins as new therapeutic strategies for preventing and treating thrombotic disorders.     

Depressed plasma activity of plasminogen or alpha2 plasmin inhibitor is not due to consumption coagulopathy in septic patients with disseminated intravascular coagulation.

We have attempted to determine whether depressed plasma plasminogen and alpha2 plasmin inhibitor (or alpha2 antiplasmin) activity is, as a result of consumption coagulopathy, a specific finding of disseminated intravascular coagulation (DIC) in septic patients. The hemostatic parameters of 139 septic patients (68 with DIC and 71 without DIC) were analyzed. Among the group as a whole, plasma activities of plasminogen and alpha2 plasmin inhibitor were significantly depressed in septic patients with DIC relative to those without DIC (P < 0.01 and P < 0.05, respectively). Notably, a significant correlation was observed between plasma levels of albumin and plasminogen activity, as well as between plasma levels of albumin and alpha2 plasmin inhibitor activity both in septic patients with DIC and those without DIC. However, no significant correlation was observed between plasma levels of plasmin-alpha2 plasmin inhibitor complex (PIC) and plasminogen activity, nor between PIC and alpha2 plasmin inhibitor activity either in septic patients with DIC or those without DIC. We concluded that depressed activity of plasminogen or alpha2 plasmin inhibitor is not as a result of consumption coagulopathy, but rather a result of low synthetic function of the liver in septic patients with DIC.     

Depletion of circulating alpha(2)-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization

In the absence of arterial recanalization, thrombolytic agents induce a dose-related extension of focal cerebral ischemic injury (FII) in experimental animals. However, FII is smaller in mice lacking alpha(2)-antiplasmin (alpha(2)-AP), the physiologic inhibitor of plasmin, suggesting its depletion might reduce FII in the absence of reperfusion. Therefore, the effect of human plasmin (Pli), human miniplasmin (mPli), and an Fab fragment neutralizing murine alpha(2)-AP (Fab-4H9) on FII after middle cerebral artery (MCA) ligation was studied in mice and in hamsters. In BALB/c mice, the median FII after 24 hours was 28 microL (range, 20-34) (n = 10) with saline and 23 microL (range, 17-26) (n = 9) with a single bolus of 0.07 mg Pli, given after MCA ligation (P =.010), which reduced alpha(2)-AP to 44% and fibrinogen from 0.75 to 0.44 g/L. FII was 20 microL (range, 13-26) (n = 6, P =.025) with 0.2 mg mPli and was 24 microL (range, 20-27) (n = 6, P =.020) with 1.7 mg Fab-4H9. Neuronal atrophy and reduction of laminin immunoreactivity were comparably observed in the infarct area after saline and Pli. In hamsters, a single bolus injection of 1 mg Pli, after MCA ligation, depleted alpha(2)-AP and fibrinogen and reduced FII at 24 hours from 20 microL (range, 9.9-38) (n = 6) to 7.0 microL (range, 0.44-31) (n = 7, P =.032). Thus, reduction of circulating alpha(2)-AP, with a single bolus of plasmin or of a neutralizing antibody fragment, significantly reduced FII after MCA ligation in mouse and hamster models, suggesting that, provided these observations can be extrapolated to human beings, transient depletion of circulating alpha(2)-AP might reduce ischemic stroke in the absence of reperfusion.     

Synergistic fibrinolysis: combined effects of plasminogen activators and an antibody that inhibits alpha 2-antiplasmin.

To improve the efficacy of plasminogen activators, we produced a monoclonal antibody (RWR) that inhibits human alpha 2-antiplasmin (alpha 2AP). In addition to inhibiting alpha 2AP in plasma, RWR binds to and inhibits fibrin cross-linked alpha 2AP and reproduces the "spontaneous" clot lysis that is the hallmark of human alpha 2AP deficiency. By inhibiting the inactivation of plasmin by alpha 2AP, RWR interacts synergistically with plasminogen activators to increase the potency (for 50% clot lysis) of urokinase by 80-fold, tissue plasminogen activator by 27-fold, and streptokinase by 20-fold. Yet, for a given amount of fibrinolysis, the combination of RWR and lower doses of plasminogen activator leads to less fibrinogen consumption than is obtained with higher, equipotent doses of plasminogen activator alone. These results suggest a strategy for increasing the efficacy of plasminogen activators. More generally, this approach to amplifying enzymatic activity by immunoneutralizing an inhibitor may be useful in other biologic processes that are rigidly governed by inhibitors.     

Roles for chloride ion and fibrinogen in the activation of [Glu1]plasminogen in human plasma.

Using two-dimensional immunoelectrophoresis and an antibody to alpha 2-antiplasmin, we assessed the plasmin generated in serum under different conditions as the plasmin-alpha 2-antiplasmin complex. Activation in serum of human [Glu1]plasminogen ([Glu1]Pg) by recombinant tissue plasminogen activator was inhibited by the normal serum levels of Cl- and was enhanced by physiological levels of fibrinogen in the presence or absence of Cl-. These results agree with the recognized ability of Cl- to induce a conformation in [Glu1]Pg less favorable for its activation than the conformation that results without Cl-. The enhancing effect of fibrinogen surpassed the inhibitory effect of Cl- over a wide range of recombinant tissue plasminogen activator concentrations in physiological serum. Lesser inhibition by Cl- was seen in a purified clot-lysis system, suggesting that [Glu1]Pg conformation when attached to soluble fibrin matrix was less affected by the anion. The data regarding the roles of circulating fibrinogen and Cl- in controlling the plasma level of activated [Glu1]Pg have important implications in thrombolytic therapy with recombinant tissue plasminogen activator.