A molecular model of plasmic degradation of crosslinked fibrin

Based on structural studies of both degrading insoluble crosslinked fibrin and of soluble derivatives, we have developed a model to explain the principal structural and physical features of plasmic degradation for crosslinked fibrin in vitro from the completely intact matrix to terminally degraded soluble derivatives. Plasmic digestion is viewed as a continuous process of proteolytic attack on accessible, enzyme-susceptible sites; but the process is separated into four phases based on solubility characteristics of the degrading fibrin matrix and on the structures of the soluble derivatives. The critical event of solubilization occurs only as the result of coincident cleavages at complementary sites in the basic two-stranded half-staggered overlap fibrin structure, resulting in the release of two-stranded complexes held together by noncovalent forces. The four smallest complexes which are released into solution have structures corresponding to DD/E, DY/YD, YY/DXD, and YXD/DXY. The protein initially solubilized has a constant composition with a predominance of large derivatives which are composed of at least on fragment from each of the two strands of the protofibril. Following their release into solution the larger complexes are converted in vitro to smaller ones by the continued action of plasmin, so that the complex found following prolonged digestion is DD/E. It is proposed that this newly defined group of complexes [11] represents the major form of circulating plasmic derivatives of crosslinked fibrin.     

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution

Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 microM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 x 10(2) M(-1) x s(-1) and a dissociation rate constant of 3.07 x 10(-4) s(-1) are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosin fibrin clot is relatively resistant to plasmin.     

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.     

Characterization of Serum Fibrinogen and Fibrin Fragments Produced during Disseminated Intravascular Coagulation

The fibrinogen and fibrin degradation products (FDP) in serum samples taken from nine patients with suspected disseminated intravascular coagulation have been characterized using a method of immunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Aall of the serum samples contained a fragment with the same electrophoretic mobility as fibrinogen fragment X, while the majority also had evidence of fragments with similar mobility to fibrinogen fragments Y and D. In eight of the nine serum samples there was strong evidence of the D-dimer fragment that is released by plasmin lysis of crosslinked fibrin. Also present in all but one of the samples were fragments of higher molecular weight than fibrinogen which were probably soluble, non-clottable, factor XIIIa induced crosslinked derivatives of fibrinogen. These results suggest that during disseminated intravascular coagulation thrombin and activated factor XIII act upon fibrin(ogen) to form complexes that are subsequently lysed by plasmin to produce soluble crosslinked derivatives of fibrin.     

Disintegration and reorganization of fibrin networks during tissue-type plasminogen activator-induced clot lysis.

In this study, we investigated tissue-type plasminogen activator (tPA)-induced lysis of glutamic acid (glu)-plasminogen-containing or lysine (lys)-plasminogen-containing thrombin-induced fibrin clots. We measured clot development and plasmin-mediated clot disintegration by thromboelastography, and used scanning electron microscopy (SEM) to document the structural changes taking place during clot formation and lysis. These events occurred in three overlapping stages, which were initiated by the addition of thrombin, resulting first in fibrin polymerization and clot network organization (Stage I). Autolytic plasmin cleavage of glu-plasminogen at lys-77 generates lys-plasminogen, exposing lysine binding sites in its kringle domains. The presence of lys-plasminogen within the thrombin-induced fibrin clot enhanced network reorganization to form thicker fibers as well as globular complexes containing fibrin and lys-plasminogen having a greater level of turbidity and a higher elastic modulus (G) than occurred with thrombin alone. Lys-plasminogen or glu-plasminogen that had been incorporated into the fibrin clot was activated to plasmin by tPA admixed with the thrombin, and led directly to clot disintegration (Stage II) concomitant with fibrin network reorganization. The onset of Stage III (clot dissolution) was signaled by a sustained secondary rise in turbidity that was due to the combined effects of lys-plasminogen presence or its conversion from glu-plasminogen, plus clot network reorganization. SEM images documented dynamic structural changes in the lysing fibrin network and showed that the secondary turbidity rise was due to extensive reorganization of severed fibrils and fibers to form wide, occasionally branched fibers. These degraded structures contributed little, if anything, to the structural integrity of the residual clot, and eventually collapsed completely during the course of progressive clot dissolution. These results provide new perspectives on the major structural events that occur in the fibrin clot matrix during fibrinolysis.     

Reactivity of D-dimer assays with the fibrinogen--fibrin split products generated by thrombolytic agents

The reactivity of two D-dimer assays (a latex agglutination method, D-Di test and an ELISA procedure, Asserachrom D-Di) to the various fibrin or fibrinogen degradation products generated in plasma by three different thrombolytic agents was analysed, in the presence or absence of a fibrin clot. Other assays performed in parallel were an ELISA assay for (DD)E complexes and the conventional fibrinogen degradation products (FDP) latex test on serum. The thrombolytic agents urokinase, streptokinase or tPA were added at various concentrations and incubated for different times ranging from 10 min to 24 h. The data showed that the D-dimer latex assay was always negative provided there was no fibrin in plasma and despite the presence of high FDP levels (greater than 600 micrograms/ml) in serum. In contrast, D-dimer or (DD)E complexes were measured by ELISA, but up to a given concentration (15-20 micrograms/ml) which reached a plateau and remained stable irrespective of the thrombolytic concentrations or the degradation times. In the presence of fibrin clot, fibrinolysis was extremely fast with tPA and the FDP were generated at a much higher concentration that that expected from the size of the fibrin clot. This suggests the existence of fibrinogenolysis targeted by the presence of fibrin but negative in its absence. Urokinase and streptokinase generated FDP very quickly but a much slower degradation rate of fibrin was observed. The immunoblotting confirmed these data and showed that no late FDP were formed in plasma even at high thrombolytic concentrations except when fibrin was present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased immunoreactivity of plasma after fibrinolytic activation in an anti-DD ELISA system. Role of soluble crosslinked fibrin polymers

After addition of a low concentration of thrombin to normal plasma, a progressive and significant increase in crosslinked fibrin polymers was found by sodium dodecyl sulfate agarose gel electrophoresis, reaching 27% of total fibrinogen and fibrin before gel formation. As measured by enzyme-linked immunosorbent assay with a monoclonal antibody specific for an epitope near the gamma gamma crosslink site, increased immunoreactivity of plasma did not occur after adding thrombin despite formation of crosslinked fibrin polymers, which indicates that the antibody does not recognize the epitope in the polymers. Addition of tissue-type plasminogen activator (t-PA) to plasma resulted in a more rapid degradation of fibrin polymers than of fibrinogen, indicating that the fibrin specificity of t-PA is retained with soluble fibrin. Coincident with degradation of plasma crosslinked fibrin polymers, plasma DD immunoreactivity increased 70-fold from 50.3 +/- 4.5 (mean +/- SD) to 3,560 +/- 1,235 ng/ml. The presence of increased crosslinked fibrin polymers produced by adding thrombin to plasma significantly increased maximum immunoreactivity after t-PA-induced degradation to 18,500 +/- 11,780 ng/ml. The increase in DD immunoreactivity was dependent on t-PA concentration; no elevation occurred below 0.01 micrograms/ml, and maximal increases occurred above 100 micrograms/ml. Analysis of gel electrophoretic patterns of thrombin and t-PA-treated plasma samples suggests that the DD reactivity of t-PA-treated plasma is mainly due to degradation of soluble crosslinked fibrin polymers. Our findings indicate that plasmic degradation of soluble fibrin polymers in plasma may be an important source of fragment DD during thrombolytic therapy.     

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ(42) specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ(42)-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ(42), and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ(42). Our results indicate the existence of 2 mechanisms of Aβ(42) involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)-fibrin binding.     

Dissolution of noncovalently bonded fibrin in the presence of bromide ions.

Two peaks were demonstrated after gel chromatography on Sepharose 6-B of noncovalently bonded firbrin dissolved in 1 M NaBr at pH 5.3. One was eluted at the void volume while the other one was eluted at the elution volume of fibrinogen. Both fractions were stable during rechromatography. The fraction eluted at the void volume was highly heterogeneous and consisted of noncovalently bonded aggregates of fibrin monomer units. Our experiments indicate that these high molecular weight derivatives develop during dissolution of fibrin in NaBr solution. The possible explanation of these results is discussed.     

The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs.

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.     

Fibrinolysis at the fluid-solid interface of thrombi

Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, a protease generated by plasminogen activators from its inactive precursor, plasminogen. Typically plasminogen activation occurs on the surface of the clot, where fibrin behaves as a cofactor in this process, and plasmin also initiates its proteolytic action at the fluid-solid interface. Although the basic reactions of the plasminogen/plasmin system in fluid phase are well characterized in terms of classical enzymology, they cannot explain completely the interfacial fibrinolytic events. Recently new methods have been introduced for quantitative evaluation of plasminogen activation on gel-phase fibrin and heterogenous-phase proteolysis, an overview of the new methodology is presented. Following formation of an interfacial lytic zone, fibrin dissolution proceeds through propagation of this zone to the core of the clot, which depends on diffusion and permeation phenomena affected by the composition of thrombi. Phospholipids (originating from platelets) form a diffusion barrier to the thrombolytic agents and also bind some of them; structural cellular proteins (namely myosin) interact with the fibrin fibers masking their cofactor and plasmin-cleavage sites. The contribution of these recent findings to our understanding of the limitations of current thrombolytic therapy is discussed. Finally, attention is focused on the termination of thrombus-associated proteolytic action in an environment abundant in proteinase inhibitors. Thus, combining together the interfacial events in the initiation, progress and termination of thrombolysis, a concept for modeling the thrombus as a temporary fibrinolytic compartment is presented.     

Plasminogen assay by means of the lysis time method.

The lysis time method for the determination of plasminogen has been investigated using plasminogen-free thrombin and fibrinogen preparations. The experiments have shown that the lysis of a fibrin clot is the result of two consecutive reactions: the formation of fibrin which proceeds as a first order reaction and the degradation of fibrin which proceeds as a zero order reaction. Plasminogen is activated in a separate reaction. If the rate of the fibrin formation is much greater than the rate of degradation, the lysis of the fibrin clot is approximately of zero order in fibrin. The lysis time will then be inversely proportional to the plasmin concentration and proportional to the fibrinogen concentration. In a double logaritmic system the correlation between lysis time and plasmin activity is a straight line with a slope of 135 degrees. Plasminogen is rapidly activated with streptokinase. Maximal activation is obtained only with a certain streptokinase concentration. Higher concentrations inactivate plasmin and with lower concentrations, the maximal activity is never reached. A spontaneous inactivation is seen after about 30 minutes. With urokinase, a higher maximal plasminogen activity is obtained than with streptokinase. Urokinase in higher concentrations does not inactivate plasmin. A standard assay for determination of plasminogen by the lysis time method has been worked out and is based on these results.     

Detection of circulating crosslinked fibrin derivatives by a heat extraction-SDS gradient gel electrophoretic technique

A technique has been developed to identify and quantitate unique plasmic degradation products of crosslinked fibrin in plasma. In this method, fibrin derivatives are extracted by heat precipitation and dissolved with disulfide bond reduction, after which the crosslinked gamma-gamma chain remnants are identified by SDS-polyacrylamide gradient gel electrophoresis and quantitated by densitometric analysis. A heterogenous group of gamma-gamma chains with molecular weights between 100,000 and 76,000 daltons was identified in lysates of crosslinked fibrin during plasmic degradation in vitro. Three stages of crosslinked fibrin degradation have been arbitrarily defined based primarily on the extent of degradation of these gamma-gamma polypeptide chains. As little as 20 microgram of crosslinked fibrin digests added to 1 ml of normal plasma could be detected by the heat-extraction--gel- electrophoresis technique, identifying the gamma-gamma derivatives with molecular weights of 96,000, 86,000, 82,000, and 76,000 daltons. Plasmic derivatives of gamma-gamma chains were not found in normal plasma, but they were identified in the plasma of patients with disseminated intravascular coagulation and deep-vein thrombosis, both before and in increased quantity during successful thrombolytic therapy.     

Fibrin subunits in venous and arterial thromboembolism.

The subunit fibrin composition of thrombi of both venous and arterial origin was examined by sodium dodecyl sulphate gel electrophoresis. The thrombi were recovered by surgical intervention and all had the same fibrin subunit composition. The alpha chains were cross-linked as alpha-chain polymers alpha (p), the gamma chains as gamma-chain dimers (gamma-gamma) while the beta chains were not crosslinked; a further subunit of molecular weight 33 000 was shown to be present in all the fibrins examined and was a degradation fragment of the beta or gamma chains. This data suggests that the crosslinked alpha chains are rate limiting to the lysis of thrombi in vivo. The digestion of pulmonary emboli by plasmin yielded soluble degradation products which were identified as D dimer and E, the latter fragments being the major products obtained by the lysis of in-vitro made plasma clots. The similarity of the composition and lysis of thrombus fibrin to that formed in vitro augurs well for the justification of in-vitro research on mechanisms in thrombolysis.     

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.     

Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator.

To explore whether fibrin fragments have binding affinity for the tissue-type plasminogen activator (t-PA) molecule, the interactions were studied of (DD)E complex and fragments DD, E1, and E3 with one-chain and two-chain t-PA. For this purpose, a solid-phase binding assay was developed using microtiter plates with nitrocellulose filters. It was found that (DD)E complex and fragments DD and E3 retained the t-PA binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin. Unexpectedly, fragment E1 did not bind t-PA. Fibrin fragments had different binding properties for one-chain and two-chain t-PA. (DD)E complex had the highest and fragment E3 the lowest affinity for one-chain t-PA, both binding curves being consistent with one class of binding sites. However, binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with fragment E3 having the highest affinity for this form of the activator. epsilon-Aminocaproic acid, even at 50 mmol/L concentration, had only minimal effect on binding of (DD)E complex or fragment DD to either one-chain or two-chain t-PA. The potentiating effect of fibrin fragments on plasminogen activation by t-PA was measured by a chromogenic substrate assay. Fragment DD was the most effective stimulator of plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory functions of fibrin, which included t-PA binding and t-PA-mediated acceleration of plasminogen activation to plasmin.     

Specific identification of fibrin polymers, fibrinogen degradation products, and crosslinked fibrin degradation products in plasma and serum with a new sensitive technique

A new method is described for identifying low concentrations of circulating derivatives of fibrinogen and fibrin, even when present in heterogeneous mixtures. This technique is applicable to plasma and serum and uses electrophoresis in 2% agarose in the presence of sodium dodecyl sulfate (SDS) followed by immunological identification of separated derivatives, using radiolabeled antifibrinogen antiserum and autoradiography. Unique electrophoretic patterns distinguish plasmic derivatives of crosslinked fibrin from those of fibrinogen and also identify crosslinked fibrin polymers produced by the combined action of thrombin and factor XIII on fibrinogen. The assay is sensitive to a concentration of 0.1 micrograms/mL of fibrinogen in serum or plasma. Fibrin polymers, plasmic degradation products of fibrinogen, and plasmic degradation products of crosslinked fibrin were detected in the plasma or serum of a patient with disseminated intravascular coagulation. Plasmic derivatives of both fibrinogen and crosslinked fibrin appeared in serum in the course of fibrinolytic therapy for pulmonary embolism, whereas during acute myocardial infarction a marked increase in the proportion of fibrin polymers in plasma was found in comparison with normal controls. Thus, the procedure can distinguish between the simultaneous processes of fibrin polymer formation, fibrinogenolysis, and fibrinolysis, and is sufficiently sensitive to detect relevant quantities of derivatives in pathologic conditions.     

Studies on the Thrombin Clotting Time

The influence on the thrombin clotting time of degradation products obtained from soluble or polymerized fibrin was studied. Plasmin degradation of soluble fibrin gave products which markedly prolonged the thrombin clotting time, whereas degradation products from polymerized fibrin (extra clot lysis) were almost inactive. The effect on the thrombin clotting time of degradation products obtained by intra clot lysis varied considerably, dependent on the relative concentrations of thrombin and plasmin used. The present observations stress the importance of a strict definition of fibrin as a source of degradation products, and are also related to the clinical interpretation of the thrombin clotting time.     

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.     

A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin.

125I-fibrinogen, adsorbed to polystyrene tubes at low ionic strength and treated with thrombin, serves as a substrate for a rapid, convenient, and sensitive test tube assay for plasmin and activators and inhibitors of this enzyme. 125I-labeled digestion products released from the 125I-fibrin-polystyrene matrix are readily separated and quantitated and behave, on gel permeation, in the same manner as plasmin-generated degradation products from an unlabeled conventional fibrin clot. The 125I-fibrin, in probable non-cross-linked form, is firmly bound to the polystyrene and is resistant to nonspecific release, with control (no enzyme) values equivalent to 15.2 ng +/- 1.2 (SD) fibrin (1% of the total bound 125I-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125I-fibrin, which exceeds published sensitivities (1000-5000 ng) using 125I- or fluorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 mug/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 mug/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/ml and 0.04 CTA U/ml, respectively (sensitivity of fibrin-plate method, 0.5 U/ml for both). The method provides a reasonable analogue of the solid-phase nature of fibrin under physiologic conditions, and the ease of preparation of large batches of tubes makes the method suitable for large-scale screening of factors modulating the plasminogen-plasmin system.