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Current therapies for posthaemorrhagic hydrocephalus (PHH) such as surgical shunting, repeated tapping and acetazolamide all have major problems and so we are exploring the possibilities of lysis of the obstructing clots. X oligomers, as indicators of fibrinolysis, were measured in CSF from 5 normal preterm infants (mean 102 ng/ml), 6 preterm infants with IVH but no PHH ( mean 315 ng/ ml) and 8 infants with progressive PHH (mean >500 ng/ ml). Serial CSF samples from one infant with IVH showed a peak X oligomer level over 1,000 ng/ml after 15 days. 4 infants were treated with repeated intraventricular injections of urokinase or streptokinase (2,500 or 5,000 units). CSF taken 2-3 days post-injection showed no change in the levels of X oligomers (already very high). Repeated doses were not associated with bleeding problems but hydrocephalus resolved in only one infant. Conclusions: There is considerable natural fibrinolytic activity in the CSF after IVH. Single intraventricular injections of modest doses of fibrinolytic agents do not have a sustained effect on CSF fibrinolysis or hydrocephalus. Larger doses by infusion look more premising.

Polyacrylamide (PA) gel electrophoresis in sodium dodecyl sulphate (SDS) has been shown to distinguish between the major components in the plasmin induced lysates of purified crosslinked fibrin and fibrinogen. The D dimer fragment from crosslinked fibrin separated on SDS-PA gels from the fibrinogen fragments X, Y, Dg and E. Follwing immuno-absorption of plasmin induced lysates of plasma clots, plasma fibrinogen, and mixtures of both, the major fragments of fibrinogen and fibrin were capable of being discriminated by SDS-PA gel electrophoresis. The presence of D dimer in patient plasma suggested the efficacy of heparin in the management of haemorrhage associated with amniotic fluid embolism.

Ten amniotic fluid samples obtained at elective caesarian section were examined for the presence of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and the two specific plasminogen activator inhibitors, PAI-1 and PAI-2. u-PA antigen was demonstrated in all samples (mean concentration equivalent to 0.35 iu/ml), but its bioactivity was very low (mean activity 0.13 iu/ml) when assessed by a bioimmuno-assay (BIA) using chromogenic substrate. None of the samples analysed by ELISA technique had measurable levels of t-PA, neither could any activity of t-PA be demonstrated by a sensitive BIA or fibrin plate technique. Significant amounts of both PAI-1 and PAI-2 antigen were present in all samples, and the mean values were 12.2 and 43.4 ng/ml, respectively. The average inhibitory capacity per ml of amniotic fluid measured by assessing the residual t-PA activity following addition of a known amount of single chain t-PA to the samples was 17.3. Thus, this study demonstrates a fibrinolytic system in amniotic fluid, which is characterised by absence of t-PA activity, low bioactivity of u-PA and excess of the two specific inhibitors, PAI-1 and PAI-2. This inhibitory capacity of amniotic fluid may play a role in preventing premature rupture of membranes.

Critically ill patients have been described as having blood coagulation abnormalities that predispose to bleeding and thrombosis. We have studied plasminogen activators, alpha 2-antiplasmin, X-oligomers fibrin fragments, fibronectin, antithrombin III, fibrinogen, platelets, kaolin-cephalin clotting time and prothrombin time on admission to the intensive care unit and sequentially after 24 and 48 hours in 39 adult patients: ARDS (n = 6), trauma (n = 12), sepsis (n = 8) and a miscellanea (n = 13). A decrease in plasminogen activators associated with an increase in X-oligomers, the earliest form of cross linked fibrin degradation products, indicate that fibrin deposition and the consumption of components of fibrinolysis is a widespread condition in the ICU patients. Low fibronectin levels were related to prognosis. These findings suggest that critically ill patients must be evaluated in respect to fibrinolysis and supported when necessary with prophylactic treatment.

Serum fibrin degradation products in a patient with severe disseminated intravascular coagulation (caused by fulminant pneumococcal sepsis), were characterized using immunoprecipitation, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and crossed immunoelectrophoresis. These revealed a spectrum of fragments identified as high molecular weight (HMW) complexes, a component with mobility on SDS PAGE similar to that of fibrinogen X (“X”), D dimer and E. By their electrophoretic characteristics and reactions with antisera to fragments E and D it was found that most of the D dimer and E were noncovalently complexed as D dimer-E, and that there was relatively little free D dimer and free E. This pattern of FDP (HMW complexes, “X” and D dimer-E) has also been identified during the lysis of crosslinked fibrin by plasmin. The HMW complexes and “X” are believed to be crosslinked X oligomers and crosslinked Y-Y or Y-D respectively.

A commercially available preparation of synthetic human fibrinopeptide B (FpB) was shown by hplc to contain two chromatographically distinct peaks, one of which was identical to FpB. Our results suggest that the contaminant peptide (FpB-2), which represented approximately 43% of the total peptide composition, is FpB containing an α-aminosuccinimide (Asc) residue. This Asc residue probably arose as a result of the cyclization of 5Asp-6Asn during either the coupling or deprotection reactions. FpB-2 was rapidly hydrolysed by carboxypeptidase B to des-Arg-FpB-2. It was stable under acidic conditions but in dilute alkali was converted to equimolar amounts of FpB and FpB containing β-Asp at residue 5. Since it has been suggested that 5Asp-6Asn is a major immunorecognition site in FpB, our observations emphasize the need to establish the purity of synthetic FpB preparations destined for use in the immunoassay of either FpB or des-Arg-FpB.

Monoclonal antibodies (mabs) were raised against X-oligomers, the earliest soluble fragments released from crosslinked fibrin (XL-FN), by the action of plasmin. Two of the mabs (NIBn 52 and NIBn 123) were monospecific for X-oligomers in that they showed no binding to fibrinogen, the plasmic fragments of fibrinogen (D and E) and non-crosslinked fibrin (X, Y, D and E), or the terminal digestion product of XL-FN, fragment DD-E. One other mab (NIBn 178) was panspecific for X-oligomers in that it exhibited a weak affinity for fibrinogen. The mabs were used to develop a two-site immunoradiometric assay (IRMA) and an enzyme-linked immunospecific assay (ELISA) which permitted the specific measurement of X-oligomers directly in plasma, rather than in serum. This immunoassay is a true assay of fibrinolysis as distinct from fibrinogenolysis and may be a potential aid in the diagnosis and evaluation of thrombosis. In preliminary studies, the assay detected low levels of X-oligomers in normal plasma and elevated levels in patients with disseminated intravascular coagulation.

SummaryA comparison has been made between the effects of pentosan polysulphate (SP54) and mucosal heparin following subcutaneous injection in man. Unlike heparin, pentosan polysulphate has relatively little effect in vivo as measured by anti-factor Xa clotting assay and none by an anti-Xa amidolytic assay (S-2222). However, pentosan polysulphate is at least as potent as heparin on a weight basis in producing activation of lipoprotein lipase, shortening of the euglobulin clot lysis time and impairing the generation of factor Xa. Our data indicate that pentosan polysulphate has more marked effects in vivo than in vitro, that the action of the drug on clotting is mediated mainly via an At III-independent pathway, and that its effects are not confined to the coagulation system.

The effect of Factor XIII-induced crosslinking of fibrin on its subsequent lysis by plasmin has been further investigated due, in part, to conflicting reports in the literature on this issue. The test system used involved 125 I labelled plasma fibrin clots and the release of the radiolabel to measure lysis rate. It was found that, while the fibrin γ-γ crosslinks do not affect the rate of fibrin lysis, the α chain crosslinkage has a significant effect. Indeed, the increased complexity of the α chain crosslinkage endows fibrin with increased resistance to lysis. 125 I labelled fibrin clots containing fibrin with and without crosslinked α chains, allowed the preferential removal of the non-crosslinked material. Discrepancies were noted when measuring the lysis of crosslinked fibrin only by chemical means (Lowry method) and by 125 I labelled peptide release. Both methods of measurement are prone to criticism; however, it was found that the 125 I release method amply reflected the release of D dimer, the major soluble structural entity in crosslinked fibrin and probably the most reliable yardstick of its lysis.

Urokinase preparations of molecular weight 55,000 (high molecular weight urokinase, HMW-UK) and 33,000 (low molecular weight urokinase, LMW-UK) were assayed against the International Reference Preparation for Urokinase (IRP-UK) by three methods. Preparations of HMW-UK and LMW-UK which had the same potency when measured by use of S-2444 differed by a factor of 2–3 when measured by clot lysis. A formula is derived which allows these differences in relative potency to be used to calculate the molar ratios of the two forms of UK in a mixture. This method was tested on IRP-UK and on four mixtures of purified HMW-UK and LMW-UK. The results of the calculations for the four mixtures compare well with their composition as prepared. The result for IRP-UK is in approximate agreement with the ratio calculated by separating this material on a Sephadex G-75 column and assaying the two forms using S-2444. The difficulty caused by the different relative activities of the two forms of UK when compared by assay methods now in use is discussed.

Carbohydrate analysis of human fibrinogen and its plasmin induced degradation products (D,E) gave molar ratios for fibrinogen of 7:18:31, for fragment D of 3:6:11 and for fragment E of 2:8:11 with respect to N- acetylneuraminic acid, N- acetylhexosamine and hexose. The ability to account for all the carbohydrate of fibrinogen following its degradation with plasmin confirms the suggested scheme of degradation in which one mole of fibrinogen produces one mole of E and two moles of D. The data are also consistent with the hypothesis that the Aα chain contains no carbohydrate, the Bβ chain contributes carbohydrate to the D fragment and the γ chain to the E fragment. The results indicate that the β chain may contain two carbohydrate side chains of 9 or 10 residues and the γ chain contains one side chain of 9 or 10 residues.

SINCE the institution of these annual lectures to the. memory of Professor E. J. Conway, FRS, some individual lecturers so honoured have had the privilege of working in Ireland at a time when the late E. J. Conway influenced greatly the scientific scene in Ireland. I recall my attendance at a meeting of the Academy where Professor Conway made a brief appearance shortly before his death. The respect and admiration for the man amongst those present was immense and was merely a reflection of what Professor Conway meant to Irish science and medicine. For myself, I have worked abroad (mostly in the U.K.) for most of my scientific career during a time period and in a subject which did not overlap with that of Professor Conway. Despite this fact, the times are numerous when colleagues have asked "did you study under Professor Conway ? this question has been asked in various parts of Europe, the U.S. and Australia. His fame reached out to all the world through his work on transport in the kidney (Conway et al, 1937; Conway et al, 1946) and the development of immunodiffusion techniques (Conway, 1957). The true scientific philosopher was expressed in his paper which deals with oceanic evolutionary notions (Conway, 1943). He lacked narrow and exclusive thinking (which thinking, unfortunately, can bedevil even the most distinguished intellects) in that his studies of the physical aspects of biology did not exclude the

Plasmin digests of fibrinogen were examined for their ability to support the ADP-induced aggregation of washed human platelets. The extent of aggregation decreased progressively during digestion, but the major loss in activity appeared to occur as fragment X was converted to fragments Y, D and E. In contrast, the thrombin clotting time and staphylococcal cell clumping titre showed immediate and marked changes during the formation of fragment X. These changes are consistent with the loss of essential residues from the C00H-terminal region of the Aα chain early in the proteolysis of fibrinogen by plasmin. The progressive decrease in platelet aggregation, and the absence of intact Aα chain in highly active fragments, however, argues against the critical involvement of the C00H-terminus of the Aα chain in the interaction of fibrinogen with platelets.

SummaryThe catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

To investigate whether vasopressin (aVP) could have a role in the regulation of coagulation and fibrinolysis during hip surgery, venous blood samples were taken for assay of FVIII:C, FVIII R:Co, vWF:Ag, fibrinopeptide A (FPA), euglobulin clot lysis time (ECLT), high molecular weight fibrin breakdown products (XL-FDP) platelet aggregation in whole blood and aVP from seven patients undergoing elective hip surgery. Samples were taken at set points over the operative period. FVIII:C increased during the operation from a geometric mean of 0.7 iU/ml pre-operatively to 1.09 iU/ml (p less than 0.05) post-operatively. vWF:Ag and FVIII R:Co rose in a similar manner. PAA (10(6)/ECLT2) rose from 12 units pre-operatively to 167 units (p less than 0.001) at prosthesis cementing, and post-operatively fell to subnormal levels. FPA increased from 13 pmol/ml to 58 pmol/ml (p less than 0.05) at prosthesis cementing, and fell to 9 pmol/ml post-operatively. Plasma XL-FDP rose from 115 ng/ml pre-operatively to 456 ng/ml at skin closure (p less than 0.05). Plasma aVP rose from 0.5 pg/ml pre-operatively to 40 pg/ml (p less than 0.01) at division of the femoral neck. There were no changes in platelet aggregation using 1.5 microM ADP. The results demonstrate activation of coagulation and fibrinolysis during the operative procedure. The mechanisms involved in these changes are complex, but the results support the hypothesis that aVP has effects on factor VIII and fibrinolysis similar to those described for abdominal surgery.

The partial (50%) digestion of plasminogen (plgn)-enriched, 125 I labelled, crosslinked fibrin (XL-FN) in a solution of streptokinase (SK) was shown to generate an array of soluble crosslinked fragments similar in molecular size (by Sepharose 4B chromatography) to those obtained when XL-FN was digested in human serum. These fragments, following chromatographic isolation, were characterised by SDS-gel electrophoresis as crosslinked X-oligomers, Y-D and D dimer fragments and a scheme for their subunit structures is presented. All these fragments were non-covalently associated with either the E domain or structures containing the E domain via the polymerisation sites bequeathed by the originating fibrin. Comparison of approximate molecular weight estimates of the X oligomers by SDS-gel electrophoresis and by Sepharose 4B chromatography suggested that crosslinking may account for 1–2 × 10 6 of the molecular weight while polymerisation sites allow molecular sizes of up to 10 × 10 6 . The relevance of these crosslinked fragments to thrombosis and DIC is discussed.

In human volunteers unfractionated heparin and a low molecular weight fraction of heparin (LMWH) caused an increase in plasma plasminogen activator (PA) which peaked at 3 hours after subcutaneous injection. Using a perfused isolated rabbit ear model the enhancement of PA activity was confirmed and was related to the anti-Xa activity of both products infused. Using a modified rabbit Wessler model for thrombus formation it was found that, when using doses of heparin and LMWH sufficient to give a 100% antithrombotic effect, antifibrinolytic drugs (eg. e-ACA and aprotinin), negated this protective effect. It is concluded that the effect of heparin and LMWH on haemostasis is mediated in part through the enhancement which these drugs have on fibrinolysis, the latter being arguably a major defence against fibrin formation during thrombosis.

Plasminogen activators (PAs) are proteases that convert plasminogen to plasmin. Plasmin, in turn, is a protease that can lyse a fibrin clot and, therefore, PAs have a primary role in fibrinolysis. Two PAs, urokinase (UK) and streptokinase (SK), have been available for therapeutic use for years. Unfortunately, both can cause systemic fibrinogenolysis and other side effects which have limited their use. Interest has focused on a different enzyme, tissue plasminogen activator (t-PA), which will cause specific clot lysis without systemic problems. The gene for t-PA has been cloned and many biotechnology firms are preparing to produce t-PA for therapeutic use. The properties and potential for therapy of t-PA are reviewed and compared to new forms of other activators, such as pro-urokinase. How the interactions of PAs and inhibitors may affect the use of PAs is also discussed.

SummaryUrokinases of different molecular weights are now commercially available. An international collaborative study (eight laboratories) has been conducted to investigate the effect of type and concentration of plasminogen on the assay of two different urokinase preparations against the International Reference Preparation (IRP). Considerable inter-laboratory variation in relative potency estimation was found, and a small effect of plasminogen concentration, independent of type, was apparent.

Raised levels of tissue fibrinolytic activity have been observed in certain malignant tissues and these may determine metastatic spread. We have investigated the fibrinolytic enzymes tissue plasminogen activator (tPA) and urokinase (UK) in 26 breast carcinomas and 13 benign breast biopsies. The extracts were analysed for overall fibrinolytic activity on fibrin plates and by fibrin-overlay zymography after electrophoresis on SDS-PAG. Supernatants of the extracts were analysed by an antigenic immunoassay (ELISA) and a functional bioimmunoassay (BIA) using polyclonal antibodies and chromogenic substrates. All tissues contained similar tPA levels. Malignant extracts contained significantly increased UK compared with benign extracts (1.60 ± 0.37 iu/ml, 0.36 ± 0.16 iu/ml; P < 0.002). Zymography showed no inhibitor complexes and UK was almost exclusively confined to malignant tissues (x2 = 5.04, P < 0.025). The results suggest malignant transformation of breast is associated with the uninhibited and significantly increased production of UK, which may be responsible for the characteristics of malignant growth.

SummaryA bioimmunoassay (BIA) for tissue plasminogen activator (t-PA) is described which depends on the binding of t-PA and its inhibitor complexes to an immobilised IgG to t-PA and the subsequent assessment of the bound t-PA using glutamic acid- plasminogen (glu-plgn) and the chromogenic substrate, S-2251. This assay indicated that neither normal plasma nor its euglobulin precipitate contain any measurable free and functionally active t-PA. The BIA was also used to measure the level of the fast acting specific t-PA inhibitor (t-PA/INH) in plasma by assessing the residual t-PA activity in the plasma euglobulin fraction (pH 5.9), following the addition of a known amount of t-PA to the plasma.

The components found in l25I-labelled cross-linked plasma fibrin lysates obtained by alternate treatments with streptokinase (SK) and plasminogen (plgn) were examined by immunological and physicochemical methods. The more rapid and complete lysis achieved with the sequence SK → plgn was reflected by the near absence of the electropho-retically stable D dimer-E complex and high molecular weight soluble aggregates ( > 3 × 105 molecular weight), while these components were present in considerable amounts during the plgn → SK regimen. An assessment of the data from the rapid and slow regimens suggested that cross-linked high molecular weight complexes were initially released from fibrin, these being rapidly degraded to the D dimer-E complex which was subsequently converted to pure D dimer and E fragments. Two forms of the D dimer-E complex are proposed, probably varying in stability according to the number of attachment sites between the D dimer and E domains.

The rapid loss in clotting activity of commercially available thrombins during storage is not reflected by their activity as measured by synthetic chromogenic substrates. It is suggested that conformational changes occurring during storage can affect the binding sites of thrombin for fibrinogen, whereas small synthetic substrates are incapable of monitoring these changes.

SummaryAn international collaborative study involving seven laboratories was undertaken to assess the suitability of a freeze- dried preparation of human plasmin to replace the current International Reference Preparation (IRP) for plasmin. Chromogenic and fibrinolytic assays were used by all participating laboratories to assess the potencies of the Proposed International Reference Preparation (PIRP) and two other freeze-dried plasmins, one of human and one of porcine originThe data suggest that the PIRP is a more suitable standard for plasmin than the IRP in that the former binds to fibrin whereas only 50% of the latter binds. The PIRP compared well to other plasmin preparations and the potency assays were independent of the assay procedure and substrate used. Degradation studies indicated that the PIRP was far more stable than the glycerol solution of the IRP, surviving for 12 months at 37° C with no significant loss in either amidolytic or fibrinolytic activity. The International Committee for Thrombosis and Haemostasis (Bergamo, 1982) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the second International Reference Preparation for Plasmin with a defined potency of 10 International Units of Plasmin per ampoule.

A new and simple approach to the sensitive assay of individual components in complex biological fluids is outlined. The principle involves a competition between soluble and polyvinyl-immobilised antigen (Ag) for a monospecific antibody (ab′) and the subsequent assessment of this competition using an 125 I-labelled second antibody (ab″). The application of the method is described for the assay of fibrinopeptide A (FPA) in human plasma and a basal level of 400–4500 pgs/ml is reported. The assay procedure requires 3–4 hours, including a Bentonite treatment of the plasma which removes fibrinogen and allows nearly 100% recovery of the FPA. The interaction of fibrinogen with the FPA antiserum allows fibrinogen-coated polyvinyl plates to be used to monitor residual ab′. These fibrinogen-coated plates can be prepared and stored for months at 4°C without any change in their ability to bind the FPA antiserum.

The development of monoclonal antibodies with a specificity for cross-linked fibrin may have a potential role in the detection and of thrombi and thrombolytic therapy. In this study, two monoclonal antibodies with a specificity for fibrin have been examined. In vitro studies have shown NIBn 123 (which has a high affinity for X-oligomer) and DD-3B6 to bind to immobilised fibrin on PVC plates as well as plasma clots which were incubated in the presence of plasma. The Km values for NIBn 123 and DD-3B6 were 1.0 × 1010/7.7 × 108 M and 2.6 × 108 M respectively. No significant binding to fibrinogen either immobilised or in solution was found. The binding of these antibodies to a human thrombus in the jugular vein of the rabbit was monitored over a 24 hour period. Preferential binding of each antibody reached a ratio of approximately 1.0 (jugular/heart) at 24 hours and an image was detected.

SummaryPentosan polysulphate causes an increase in plasminogen activator activity in plasma both after oral ingestion and after subcutaneous injection. The effect is greatest after 3 h and has disappeared by 6 h. Repeat doses by mouth over 5 days elicit a similar response. The recorded increase in activity is due largely to the release of tissue-type plasminogen activator (tPA) from the endothelium according to the antigen assay although there could be a small contribution from Factor XH-related “intrinsic” fibrinolysis induced in vitro. SP54 enhances activity ex vivo by a non-specific surface effect, and this phenomenon may contribute the increased levels of activity seen in vitro. Administration of SP54 to animals elicits a similar increase in activator activity, the intramuscular route being slightly more effective. Results with an inferior vena cava thrombosis model in the rat suggest that pentosan polysulphate may induce a thrombolytic effect.

The fate of the peptide-like links induced in a fibrin polymer by Factor XIII and Ca2+ was examined following plasmin (EC 3.4.4.14) degradation of crosslinked fibrins. The γ ∗ - chain crosslinks (forming γ-γ dimers in fibrin) were shown to be conserved in the D core fragment giving rise to D dimers which were crosslinked via the D (γ) g chains. It seems that none of the crosslinks survive in the other well known fibrinogen core fragment E. These results suggest that the α chain crosslinks (which give rise to a α polymers in crosslinked fibrin) are removed from crosslinked fibrin by plasmin attack. Since the D-like core fragment of crosslinked fibrin is really a dimer of D (mol. wt about 164 000) it may be readily confused with the early degradation fragments of fibrinogen (X and Y) and could further confuse the distinction between fibrinogen and fibrin degradation products.

Summary 1. Human serum cholinesterase (EC 3.1.1.8) was purified about 500 times from reconstituted outdated plasma by (NH4)2SO4 acid precipitation and chromatography on DEAE-cellulose. The yield was 45%. 2. The enzyme system showed seven major components on polyacrylamide gel electrophoresis, six of which survived the purification procedure and these were reduced in number to asingle rather diffuse component following prolonged treatment with neuraminidase (EC 3.2.1.18). Gel electrophoresis suggested that the five fastest components differed in size, but that aggregation of the fastest component to give different polymers of the enzyme was not a reasonable explanation of the heterogeneity. N-Acetylneuraminic acid plays an important role in maintaining the stability of this multicomponent system. 3. A tentative suggestion for the gross structure of the pseudocholinesterase molecule has been suggested.

SummaryAn international collaborative study involving seven laboratories was undertaken to assess which of three lyophilised preparations might serve as an International Standard (I.S.) for tissue plasminogen activator (t-PA). Two of the preparations were isolates from human melanoma cell cultures while one was of pig heart origin. A clot lysis assay was used by all participants in the study.The data suggested that both preparations of human cell origin were comparable, in that their log dose-response lines were parallel, while that of the porcine preparation was not. Accelerated degradation studies indicated that one melanoma extract (denoted 83/517) was more stable than the other and it was decided to recommend preparation 83/517 as the standard for t-PA. The International Committee for Thrombosis and Haemostasis (Stockholm 1983) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the International, Standard for tissue plasminogen activator, with an assigned potency of 1000 International Units per ampoule.

SummaryA urokinase-activated plasmin (UK-plasmin) preparation was assayed against the International Reference Preparation for Plasmin (IRP-plasmin) using caseinolytic, fibrinolytic, fibrinogenolytic and chromogenic assay methods. The relative potency (using multi-dose bioassays) was estimated by the fibrinolytic method to be about twice that obtained by the caseinolytic, fibrinolytic and chromogenic assay methods. It was found that the UK-plasmin binds to fibrin to a greater extent than does the IRP-plasmin and this is advanced as an explanation for the discrepancy between assay methods. This difference in the binding of the two plasmins to fibrin may mean that it will be difficult to compare the fibrinolytic activities of various plasmin preparations.It is also shown that, during thermal degradation, the IRP-plasmin loses fibrinolytic activity more rapidly than amidolytic activity.

SummaryThe effect of strenuous exercise on the fibrinolytic and coagulation mechanisms was examined in six healthy male subjects. Five min bicycle exercise at a work-rate of 800 to 1200 kpm. min−1 produced an abrupt increase in plasma plasminogen activator levels which disappeared after 90 min. However, there was no change in early or late fibrin degradation products nor was there a change in fibrinopeptide A levels or βthromboglobulin levels after exercise although activated partial thromboplastin times were significantly shortened. It is concluded that strenuous exercise does not produce any real increase in fibrinogen-fibrin conversion nor any real increase in the breakdown of these proteins. The role of exercise-induced release of plasminogen activator remains unclear, but probably helps to maintain plasma levels in a discontinuous manner concurrently with the continuous low-level secretion from the vascular wall. The shortening of partial thromboplastin time may be due to the raised levels of plasminogen activator changing the activation state of other coagulation factors.

Physicochemical and immunological comparisons between plasmin mediated crosslinked (XL) fibrin digests made in buffer, serum and buffered Trasylol systems have shown that all the D dimer released from XL fibrin is associated with the fibrin E fragment in a strong, non-covalent and electrophoretically stable complex which has the empirical formula, 2D-E. The complex dissociates and reassociates in the presence and absence of detergent. The subunits of D dimer moiety of the complex are made up of Aα chain remnants (MW 12, 000), Bβ chain remnants (42,000) and crosslinked γ chain remnants (MW about 80, 000). A diagram showing the locations of the three domains of the complex in three distinct fibrin subunits is presented. Aggregates of the complex were found in fibrin lysates made in human serum and their significance to fibrin lysis in vivo is discussed.

Synthetic tripeptides (code named S-2160 and Chromozym TH) have been used as substrates for the measurement of thrombin activity in two commercial preparations of bovine thrombin, using the International Standard for thrombin (I.S. thrombin) for comparison. The results, when compared to the data obtained using the natural substrate, fibrinogen, indicated the following: 1) the thrombin potency of one commercial thrombin was much higher using the synthetic substrate assays than when the clotting assay was used; the synthetic substrate data for the other commercial thrombin differed significantly from the clotting assay data but in a less consistent manner. 2) the loss of the biological activity of thrombin when stored in frozen aliquots (−20°C) was very rapid, while the amidolytic activity of similarly stored samples did not alter significantly. It was concluded that the synthetic substrates used in this study were of little use when measuring the biological activity of thrombin preparations in comparison to the established standard for thrombin (I.S. thrombin).

This is a brief review of the major events which take place when plasmin reacts with fibrinogen (fibrinogenolysis) and fibrin (fibrinolysis). An effort is made to interrelate the locations of the major products of fibrinogenolysis (X, Y, D and E) in the originating fibrinogen molecule. The complexity of the lysates of fibrins, cross-linked by factor XIII to varying extents, is discussed. Clinical and structural implications of two fragments (D dimer and the D dimer-E complex) unique to cross-linked fibrin digests, are highlighted.