Binding of various thrombin fractions to fibrin and the influence of AT-III on their adsorption.

Human thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent alpha-thrombin, 28 per cent beta-thrombin and only 2 per cent gamma-thrombin. Although alpha-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved. High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin. To avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/l. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration. Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/l. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin. Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/l). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Venous thrombosis following diagnostic transvenous catheterization by percutaneous catheter insertion: an evaluation of desmopressin as a thromboprophylactic agent.

The preventive effect of desmopressin with respect to catheter induced thrombosis was studied in a randomized double-blind trial, consisting of 30 patients undergoing percutaneous transcubital right heart catheterization. Phlebography of the catheterized arm was performed after five days. The frequency of post-catheterization thrombosis was reduced by 33 per cent, from 86 per cent in the treatment group to 53 per cent in the control group (0.1 less than p less than 0.2). This effect was restricted to minor thrombi, whereas major thrombosis could not be prevented. Patient materials such as that of the present study, may become useful in preliminary investigations of thromboprophylactic agents.

Thrombus-related uptake and vascular clearance of 131 I-fibrin des-AABB as compared to 125 I-fibrinogen in patients with established venous thrombosis.

Thrombus-related uptake of 131 I-fibrin des-AABB has been compared to that of 125 I-fibrinogen in 13 patients with established venous thrombosis. Both tracers originated from a common pool of beta-alanine precipitated fibrinogen. Scan-recordings were performed as a radiofibrin (ogen) uptake test. Uptake characteristics of des-AABB fibrin were similar to those of fibrinogen, when measured as percentage of concomitant radioactivity over the heart. Due to its longer circulation time, fibrinogen was superior to fibrin des-AABB for the detection of venous thrombi. Circulating des-AABB fibrin was cleared biphasically, with an initial rapid decline followed by a gradual exponential decrease. Mean half-lives were 5.5 +/- SD 3.5 hr and 10 +/- SD 3.5 hr, respectively. The elimination rates were uninfluenced by thrombus activity, as judged by the fibrin(ogen) uptake test. Metabolic half-life of fibrinogen in the total material was 62 +/- SD 19 hr. Dissociation of fibrinogen and soluble des-AABB fibrin clearance rates was evident, describing their own, independent elimination patterns, probably reflecting different clearing mechanisms.

Functional and antigenic antithrombin III values in plasma and serum from 115 regular blood donors and antithrombin III in prediction of postoperative thrombosis.

Antithrombin III (AT-III) was determined functionally using chromogenic substrate (S-2238) and immunologically using radial immunodiffusion (RID) in plasma and serum from 115 blood donors. There was a decrease in functional activity by 43.5% and in antigen concentration by 18.4% during in vitro coagulation when corresponding serum and plasma samples were compared. A positive correlation was found between the two methods in plasma (r = 0.784) as well as in serum ( r = 0.658). RID in serum correlated well with RID in plasma (r = 0.811), but the correlation for S-2238 in plasma and serum was poor (r = 0.411). Functional AT-III decrease during coagulation was uncorrelated with age and fibrinogen, and was not statistically affected by sex, smoking or blood groups. Preoperative functional AT-III activity was measured in 25 patients undergoing elective hip replacement, 60% of whom developed postoperative thrombosis. The functional activity in serum discriminated well between the thrombotic and nonthrombotic group of patients (p less than 0.025), whereas the activity in plasma showed only a minor difference.

Comparison of three D-dimer assays for the diagnosis of DVT:ELISA, latex and an immunofiltration assay (NycoCard D-Dimer).

Ninety-two consecutive patients referred for suspicion of deep venous thrombosis (DVT) were analyzed for D-dimer using ELISA, latex test, and a new immunofiltration method (NycoCard D-Dimer). Contrast venography verified the diagnosis in 40, and excluded the diagnosis in 52 patients. The sensitivity, negative predictive values, specificity and positive predictive values were, for ELISA 98%, 95%, 38% and 54, for NycoCard D-Dimer 100%, 100%, 42% and 57% and for the latex test 73%, 78%, 75%, and 69%, respectively. Sensitivity and specificity were inversely related with increasing pathological cut-off value. Comparison of test results by concentration category revealed a good agreement between ELISA and NycoCard D-Dimer, but to less extent between latex and the two other tests. It is concluded that NycoCard D-Dimer and D-dimer ELISA are well-suited as exclusion tests for DVT. A plasma sample is tested with NycoCard D-Dimer in less than 2 min. Thus, this test combines advantageous analytical properties comparable to the ELISA-test, with rapidity and simplicity comparable to the latex test.

Quantitation of the three normally-occurring plasma fibrinogens in health and during so-called "acute phase" by SDS electrophoresis of fibrin obtained from EDTA-plasma.

Normal human plasma contains fibrinogens of different molecular weight. To quantitate these fibrinogens, plasma was clotted at pH 6.4 with 7.5 NIH U/ml thrombin in the presence of 8 mM EDTA. The clots were dissolved in 6.6 M urea and submitted to SDS electrophoresis on gels containing 3% polyacrylamide/0.5% agarose, and the fractions quantitated by densitometric scanning. The reproducibility of this method was high with variation coefficient 1.5%. Three main fibrinogen fractions were found: High molecular weight fibrinogen (HMW, mw 340 000), low molecular weight fibrinogen (LMW, mw 300 000) and LMW' (mw 280 000). In addition 5 weak bands could be seen. In plasma from 123 healthy subjects of both sexes, aged 1-93 years, HMW constituted 69.7% +/- 5.1, LMW 26.5% +/- 4.8 and LMW' 3.8% +/- 1.8 of the total fibrinogen. Women and older subjects displayed a small, but statistically significant reduction in the relative amounts of HMW (2-4%). Following surgery and extensive acute myocardial infarction the HMW/LMW ratio changed. The substantial increase in total fibrinogen regularly recorded was mainly due to HMW that reached maximal values after 3-4 days. LMW remained unchanged the first 2 days and then displayed a slight increase with a delayed maximum (8-11 days).

Evidence that changes in fibrinogen quality during acute phase reactions are of major importance for the amount of heparin precipitable fraction (HPF).

The influence of qualitative and quantitative changes in plasma fibrinogen upon the amount of heparin precipitable fraction (HPF), obtained during the course of acute disease states, were examined. Whereas fibrinogen in plasma includes 3 species of different molecular weights (HMW, LMW, LMW'), fibrinogen in HPF, from normal as well as from "acute phase" plasma, consisted almost exclusively of HMW-fibrinogen (3% SDS-PAGE). Sub-unit chain electrophoresis (10% PAGE-SDS and two-dimensional electrophoresis) did not disclose any signs of abnormalities in the fibrinogen precipitated in HPF. Studies on patients revealed that the composition of plasma fibrinogen changed (increased HMW) during the course of acute myocardial infarction and following hip operations, and indicated that the amount of HPF was governed by the relative amount of HMW-fibrinogen as well total fibrinogen. This assumption was strengthened by quantitating the amount of HPF obtained after addition of highly purified HMW and LMW fibrinogen to normal and "acute phase" plasma. It is concluded that fibrinogen quality (per cent HMW or HMW/LMW ratio) is of major importance for the amount of HPF, thereby explaining some of the HPF-variations seen during the course of acute diseases.

Inhibition or acceleration of fibrin polymerization by monoclonal immunoglobulins and immunoglobulin fragments.

A prolongation of the thrombin clotting time by at least 25% was found in the presence of 8 of 26 IgG myeloma proteins, 2 of 5 IgA proteins and 1 of 10 macroglobulins, in a concentration of 10 mg/ml. Normal pooled immunoglobulin in concentrations up to 83 mg/ml were inactive. 2 IgG and 2 IgM monoclonal immunoglobulins caused an acceleration of the thrombin time by at least 20%; this phenomenon has not been reported before. Corresponding results were obtained with these proteins when the polymerization of des-AA and des-AABB fibrin monomers were investigated in a light scattering system. Studies with enzymatically produced Fab, F(ab')2 and Fc fragments from normal and monoclonal immunoglobulin suggested that the polymerization inhibitory activity resides in the Fab portion of the immunoglobulin molecule. While the ability to inhibit fibrin polymerization may be an immunoglobulin specific effect, the acceleration caused by certain other monoclonal immunoglobulins may be an unspecific effect similar to that seen with dextran or albumin.

Does qualitatively altered fibrinogen contribute to the increased heparin precipitable fraction (HPF) in acute myocardial infarction (AMI)?

The amount and protein composition of heparin precipitable fraction (HPF), and the plasma concentrations of fibrinogen, fibronectin, alpha 1-acid glycoprotein, alpha 1-antitrypsin, haptoglobin and C-reactive protein (CRP) were determined in healthy subjects as well as during the course of acute myocardial infarction (AMI). In all samples tested, HPF consisted nearly exclusively of fibrinogen and fibronectin. In the small precipitates from healthy subjects, approximately equal amounts of these two proteins were recovered. During the first days of AMI, plasma fibrinogen increased 2-3 fold. At the same time, however, the amount of HPF increased 5-10 fold. This increase was associated with increased precipitation of fibrinogen, whereas no simultaneous increase in fibronectin was found. In fact, a slight drop in plasma as well as HPF-fibronectin was regularly observed during this period. During the following week HPF returned to normal values, whereas the plasma fibrinogen remained elevated. No satisfactory explanation for this discrepancy can be offered at present. Thus, no evidence could be provided that other acute phase proteins than fibrinogen itself were involved. Some experiments, however, indicated qualitative changes in the fibrinogen participating in the HPF precipitation. Further studies are necessary to clarify this point. Such studies are in progress.

The release of small amounts of fibrinopeptide-B (FPB) is of critical importance for the thrombin clotting time.

When fibrinogen is clotted with thrombin, mainly fibrinopeptide-A (FPA) is released at gel point, the FPB-release being delayed. The present study present evidence that the small amounts of des-AABB fibrin found at visible gelation improves polymerization, thereby determining the thrombin clotting time. 1. Purified fibrinogen was clotted with thrombin or Reptilase, peptide release monitored radioimmunologically. With thrombin, significantly less fibrin (released FPA) was necessary for gel formation than when Reptilase was used. Furthermore, approximately 10% of the available FPB had been released by thrombin at gel point. 2. Polymerization of monomers prepared by incubation of fibrinogen with Reptilase (des-AA) and thrombin (des-AABB) in urea, were examined by light scattering. Des-AA fibrin monomers polymerised after a lag-phase of 4 minutes. When 20% of these monomers were substituted with des-AABB monomers, the lag was reduced to 1 1/2 minutes. 3. Finally, polymerization of preformed des-AA monomers was studied with and without the addition of small amounts of thrombin, and the FPB-release monitored. The presence of 0.04 NIH U/ml of thrombin reduced the lag from 10 minutes to 3 minutes. At this time approximately 10% of FPB had been released. It is concluded that even small amounts of des-AABB fibrin substantially improves polymerization of des-AA fibrin.

The stimulatory effect of soluble fibrin on plasminogen activation by tissue plasminogen activator as studied by the Coa-set Fibrin Monomer test.

The stimulatory effect of various fibrin preparations on plasminogen activation by tissue plasminogen activator, was studied by the Coa-set Fibrin Monomer test (Kabi). Fibrin obtained by complete conversion of purified fibrinogen demonstrated a greater stimulatory effect on plasminogen activation than did equal amounts of fibrin obtained by partial conversion of fibrinogen. Soluble fibrin generated by treating human plasma with minute amounts of thrombin or bathroxobin, resembled partially converted purified fibrinogen. The plasminogen activating effect of completely converted fibrinogen was similar in thrombin and bathroxobin incubated samples. In preparations of partially converted fibrinogen and in plasma samples, bathroxobin digested fibrinogen expressed a more pronounced stimulatory effect on plasminogen activation than did thrombin digested specimens. The underlying mechanism for these differences are discussed.

Half life and incorporation into venous thrombi of the normally occurring plasma fibrinogen fractions HMW and LMW.

The main plasma fibrinogen species, high molecular weight fibrinogen (HMW, mw. 340,000) and LMW (mw. 305,000), displaying different in vitro properties, were examined as to half-life and incorporation into venous thrombi (DVT). Total plasma fibrinogen and relative amounts of HMW were measured pre- and postoperatively in eighteen patients undergoing total hip arthroplasty, and related to the occurrence of deep vein thrombosis, as determined by the fibrinogen uptake test (FUT). Total fibrinogen and HMW did not disclose significant differences between scan-negative and scan-positive groups. HMW and LMW, prepared from purified fibrinogen, were labelled with I125 and I131, injected simultaneously and the incorporation into thrombi registered by leg-scanning. In 5 patients demonstrating a positive FUT, HMW as well as LMW were incorporated approximately to the same extent. This result implies that neither of these fibrinogen fractions offer any advantage as compared to ordinary fibrinogen when used for FUT. The half-lives of HMW and LMW were calculated from the elimination curves of the plasma clot-radioactivity. In all the surgical patients (n = 10) as well as in the two medical DVT-patients and in two healthy volunteers the half-life of LMW was approximately 10% longer than that of HMW.

Thrombin treated plasma employed as a standard for determination of soluble fibrin.

The Coa-set Fibrin Monomer test (CFM-test) is a quantitative method for determination of soluble fibrin in plasma. The fibrin standard of the CFM-test is produced by bathroxobin conversion of purified fibrinogen to fibrin. Plasma treated with minute amounts of thrombin may be considered as a more physiological way of preparing fibrin monomers. Therefore, we have employed such thrombin treated plasma (TTP) as an alternative fibrin standard for the CFM-test. The fibrin concentration of the TTP was determined indirectly by quantitation of released fibrinopeptide A. The TTP was stable during freezing, thawing and storage for 3 months at -70 degrees C. The standard curve obtained using TTP as a standard was linear in the range of 0-275 nmol/l fibrin in plasma, but the slope of the line was less steep than the original standard curve. This difference was probably due to the greater plasminogen activating effect of bathroxobin digested fibrinogen compared to soluble fibrin generated in plasma by thrombin, as observed in a previous study. Because of the less steep slope of the alternative standard curve, fibrin levels in plasma samples from 20 healthy volunteers and 25 patients were found to be higher employing TTP as a standard. Preparation of a fibrin standard by incubation of plasma with minute amounts of thrombin will to some extent mimic the process of fibrin generation in vivo. Since we have found a satisfactory stability of such a standard during freezing, thawing and storage, we think the TTP standard might be useful for quantitation of soluble fibrin in plasma.

Quantitation of factor XIII by SDS polyacrylamide gel electrophoresis.

An electrophoretic method for the determination of f.XIII, based on the capability of f.XIIIa to cross-link fibrin clots (5) was studied, and the great sensitivity of the method confirmed. Thus, with suitable technical conditions, f.XIII activities less than 0.1% of that in normal human plasma could be detected. The results obtained in plasma from various patients and in f.XIII concentrates (cryoprecipitates) corresponded well with those obtained with the dansylcadaverine method (3) and with the urea solubility test, with two exceptions: In a patient with severe congenital f.XIII deficiency, only the present method was sensitive enough to detect any f.XIII-activity (about 0.1%), and in a patient with an inhibitor against f.XIIIa, the dansylcadaverine method failed to detect this. The present method is too laborious for routine screening, but is recommended as an alternative references method. It may prove especially suitable to detect minute amounts of f.XIII.

Comparison of methods for detecting soluble fibrin in plasma. An in vitro study.

The ability of the COA-SET Fibrin monomer (COA-SET FM) test to detect soluble fibrin was evaluated by comparing the results of the COA-SET FM test with fibrinopeptide A (FPA) determinations following thrombin incubation of plasma or whole blood. In addition, two semiquantitative tests (erythrocytes-agglutination test (FM-test) and ethanol gelation test (EGT] were included in the study. Under the experimental conditions used, the COA-SET FM test proved less sensitive than the FPA-assay. There was a strong correlation between the results obtained by the two tests (r = 0.86, p = 0.0001). When solely regarding low levels of soluble fibrin, however, the correlation was weaker (r = 0.59, p = 0.0003). The FM-test was less sensitive than the COA-SET FM test, but more sensitive than EGT at normal and low fibrinogen concentrations. At high fibrinogen concentrations, however, EGT proved more sensitive than the FM-test. Knowing that 1-2 moles of FPA are released per mole of fibrin monomers formed, a discrepancy was observed between the FPA concentrations and the fibrin monomer concentrations as determined by the COA-SET FM test, the FPA levels being 2-25 times higher than the fibrin monomer levels. The discrepancy was greatest at incipient fibrinogen-fibrin transformation and at high plasma fibrinogen levels. This may suggest that fibrinogen in some way interfered with the stimulating effect of fibrin on the t-PA catalyzed activation of plasminogen, the principle upon which the COA-SET FM test is based.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of a heat treated antihaemophilic cryoprecipitate.

In order to evaluate the influence of heat treatment (68 degrees C for 24 or 72 hours) on the essential components of antihaemophilic cryoprecipitate, i.e. factor VIII coagulant activity (VIII:C), von Willebrand factor (VIIIR:Ag and VIIIR:RCF) and fibrinogen, ordinary lyophilized cryoprecipitate was compared to heat treated, aminoacid-enriched specimens. The median reduction in factors VIII:C, VIIIR:Ag, VIIIR:RCF and fibrinogen during lyophilization of ordinary cryoprecipitate was 26 per cent, 11 per cent, 1 per cent and 8.5 per cent, respectively. Heat treatment of such cryoprecipitate resulted in 85 to 98.5 per cent reduction in these parameters, while the reduction following lyophilization and heat treatment (24 hours) of aminoacid-containing preparations was not significantly different from non-heated, ordinary cryoprecipitate. Following heating of aminoacid-enriched cryoprecipitate for 72 hours, only factor VIIIR:RCF was significantly reduced (32.5 per cent) compared to non-heated samples. Ordinary cryoprecipitate was almost insoluble following heat treatment. Enrichment with aminoacids, however, made the heat treated cryoprecipitate fully soluble, but the content of these vials were slightly slower in dissolving than non-heated preparations. Ultracentrifugation prior to lyophilization and heating did not improve the solubility. If heat treatment proves to be efficient in inactivating viral agents, we conclude that heated (68 degrees C for 24 hours), aminoacid-enriched cryoprecipitate may be a convenient product for treating haemophilia A, von Willebrand's disease and hypofibrinogenemia.

Polymerization properties of two normally circulating fibrinogens, HMW and LMW. Evidence that the COOH-terminal end of the a-chain is of importance for fibrin polymerization.

The plasma fibrinogen fractions HMW (mw 340,000) and LMW (mw 305,000) were prepared from purified (beta-alanine precipitated) fibrinogen by step-wise precipitation with ammonium sulfate. The thrombin clotting times were 14" and 20" respectively. The enzymatic phase of coagulation, measured as release of fibrinopeptide-A during incubation with thrombin, was found to be identical for HMW and LMW. Polymerization was studied by light scattering (at 605 nm) using preformed monomers (des-AA and des-AABB) prepared from HMW and LMW in the presence of 3.3 M urea by incubation with thrombin (100 NIH U/ml final conc.) and reptilase (1 U/ml final conc.). The HMW-monomers polymerised at a substantially higher rate than the corresponding LMW-monomers. Thus, the prolonged clotting time of LMW was explained by retarded polymerization. It is suggested that the -COOH terminal end of the a-chain, containing the molecular difference between HMW and LMW, is of importance for polymerization. Furthermore, the release of fibrinopeptide B (des-AABB-monomers) improved polymerization properties in HMW as well as in LMW, and all types of monomers polymerised more rapidly in the presence of Ca++.

The molecular localization of the ability of certain monoclonal immunoglobulins to interfere with fibrin polymerization.

When a purified myeloma protein with the ability to inhibit fibrin polymerization was preincubated with sheep anti-human F(ab')2 antibodies, there was a marked reduction of the ability to prolong the thrombin clotting time, while anti-Fc antibodies had only a slight effect. A monoclonal immunoglobulin which shortens the thrombin time was uninfluenced by anti-F (ab')2 as well as by anti-Fc antibodies. Partial reduction and alkylation of the immunoglobulins did not modify their effect on the thrombin time. None of the immunoglobulins bound to fibrinogen or fibrin monomer that had been linked to CNBr-activated agarose. Myeloma proteins may interfere with fibrin polymerization in two ways: some proteins inhibit polymerization in a reaction that depends on the Fab (antigen binding) part of the molecule; however, antibody binding activity against fibrinogen or fibrin monomer has not been demonstrated. Other (rare) myeloma proteins accelerate fibrin polymerization; this effect cannot be ascribed to any particular portion of the molecule and is probably of an unspecific nature.

Purification and characterization of 3 fibrinogens with different molecular weights obtained from normal human plasma.

Fibrinogen was purified from fresh citrated human plasma by precipitation with beta-alanine in the presence of citrate and protease inhibitors. From this material, fractions corresponding to the HMW (high molecular weight), LMW and LMW' fibrinogen fractions of plasma were obtained by step-wise precipitation with ammonium sulfate. Electrophoresis revealed that HMW was contaminated with 4% LMW, LMW was contaminated with 6% HMW, and the LMW'-fraction was a mixture of LMW' (50%), LMW (20%) and two derivatives of intermediate m.w.. The HMW fraction (mw. 340 000) contained intact Aa-chains, while the molecular weight of LMW was reduced to 305 000 and that of LMW' to 270 000 due to proteolysis of the -COOH terminal end of one (LMW) or both (LMW') Aa-chains. The clottability of HMW was 98%, of LMW 92% and of LMW' about 80%. Thrombin clotting times (1 NIH U/ml) were 14", 20" and 25" resp. These differences were highly accentuated when clotting was performed with reptilase (14", 38" and 120"). Contamination with soluble fibrin was less than 2% and the contents of fibronectin and AT-III were low. No thrombin or plasmin activity was generated upon 24 hours incubation at +4 degrees C as evidenced by increased content of fibrinopeptide-A and fragment Bb-15-42 resp.

Degradation of fibrinogen and cross-linked fibrin by human neutrophil elastase generates D-like fragments detected by ELISA but not latex D-dimer test.

Recently, we observed that D-dimers are degraded by human neutrophil elastase (HNE) into two D-like fragments, reacting with the monoclonal antibody in an ELISA D-dimer test but not reacting with the corresponding latex D-dimer test. To investigate this in more detail, we studied the degradation of cross-linked fibrin and fibrinogen by plasmin and HNE to see if this resulted in D-fragments or D-like fragments. Degradation of fibrinogen, both by plasmin and HNE, resulted in D- and D-like fragments, respectively, detected by the ELISA D-dimer test. Degradation of cross-linked fibrin by plasmin and HNE also resulted in D- and D-like fragments, which were detected by the ELISA method. Intact D-dimers detected by the latex D-dimer test were only observed after degradation of cross-linked fibrin with plasmin. We conclude that during lysis of cross-linked fibrin as well as fibrinogen by plasmin and HNE, D-fragments, and D-like fragments, detected by the ELISA D-dimer test, are formed. Only during degradation of cross-linked fibrin by plasmin, intact D-dimers, detected by latex D-dimer test, are formed. The ELISA D-dimer test may therefore be used to detect fibrin and fibrinogen degradation products generated by the combined action of plasmin and HNE in sepsis and other conditions with increased HNE activity, while the latex D-dimer test may be used to detect plasmin derived intact D-dimers.