The effect of alcohol ingestion on the exercise-induced changes in fibrin and fibrinogen degradation products in man.

The present study examined the influence of ingesting a moderate dose of alcohol on plasminogen activator activity (t-PA), plasma fibrinogen (Fb), total degradation products (TDP) and the degradation products of fibrin (FbDP) and fibrinogen (FgDP) at rest and in response to exercise. Eleven male subjects performed two separate experimental trials at an exercise intensity corresponding to 70% maximal oxygen consumption for 35 min. Prior to trials, subjects were either given 0.5 g/kg alcohol in orange-flavoured drink or an equal volume of non-caloric non-alcoholic drink 45 min before exercise. Comparison of the levels of t-PA, Fb, TDP, FbDP, and FgDP at rest, before and 45 min after the ingestion of alcohol revealed no significant differences between alcohol and control experiments. Exercise resulted in a marked increase in t-PA, TDP, and FgDP, with no appreciable change in FbDP. Although plasma fibrinogen level showed significant decrease post-exercise when subjects ingested alcohol, this difference was small and its biological significance is questionable. While t-PA level increased similarly in response to exercise during alcohol and control trials, a significantly higher response of TDP was found during the control trial compared with alcohol trial. It was concluded that exercise with and without alcohol ingestion is followed by a substantial increase in t-PA, which coincided with an increase in TDP. The increase in TDP was mainly due to an increase in FgDP, but not to FbDP. These findings support the hypothesis that a significant fibrinogenolysis occurs in response to exercise, and moderate intoxication with alcohol prior to exercise reduced this response.     

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

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

Thrombin activation and increased fibrinolysis in patients with chronic liver disease

The respective roles of intravascular coagulation (DIC) and fibrinolysis were assessed in severe chronic liver disease by measuring thrombin-antithrombin (TAT) complexes, tissue-type plasminogen activator antigen (tPA Ag) and fibrinogen and fibrin degradation products (FgDP and FbDP respectively) in 66 patients with liver disease caused by cirrhosis (n = 34) or chronic hepatitis (n = 32) as compared to findings in a control group (n = 30). There was a significant increase of TAT complexes (P less than 0.01), tPA Ag (P less than 0.002), FDP and FbDP (P less than 0.001) in patients as compared to controls. FbDP increase was more evident in patients with cirrhosis than in those with hepatitis (P less than 0.01). Significant correlations between these parameters with some liver function tests were also demonstrated. Thus, in patients with severe liver disease, an increased thrombin activity, as demonstrated by high TAT levels; followed by hyperfibrinolysis suggest that a low grade DIC may occur.     

Fibrin degradation products generation and fibrinopeptide A release in normal plasma incubated with thrombolytic agents: proposed mechanisms.

Clinical data have shown that the evaluation of fibrin degradation products (FbDP) does not reflect the efficiency of thrombolytic therapy in vivo. In this study, we found that the addition of plasminogen activators to normal plasma resulted in generation of FbDP and release of fibrinopeptide A (FpA) as shown by ELISA and HPLC. This FpA release was concomitant with fibrinogen degradation, and was not inhibited by thrombin inhibition or by prothrombin depletion in plasma. Thus, the increase in FpA did not result from coagulation activation and may result from the plasmin-induced release of FpA from fibrinogen degradation product E1. The generation of cross-linked FbDP after tPA addition occurred in normal plasma as well as in factor-XIII-deficient plasma and quickly reached a plateau. It was not inhibited by hirudin. Therefore FbDP in these plasmas probably derived from the plasmin degradation of cellular transglutaminase cross-linked fibrin/fibrinogen derivatives present in plasma.     

Fibrinolysis and fibrinogenolysis in patients with thrombotic disease.

In order to assess the fibrinolytic state in thrombotic disease, plasma levels of fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) were measured in 126 patients with a variety of thrombotic diseases. Mean plasma concentrations of both FbDP and FgDP were significantly elevated in patients with thrombotic disease as compared with healthy subjects. Plasma concentrations of FgDP were positively correlated with FbDP (r = 0.667, P less than 0.001). When analysed according to the disease categories, the magnitude of elevations of FbDP and FgDP was most prominent in venous thrombotic disease such as deep vein thrombosis and pulmonary embolism. These findings indicate that fibrinolysis is accelerated in patients with thrombotic disease and that fibrinolysis is frequently accompanied by some fibrinogenolysis in these patients.     

Analysis of fibrin formation and proteolysis during intravenous administration of ancrod

Ancrod is a purified fraction of venom from the Malayan pit viper, Calloselasma rhodostoma, currently under investigation for treatment of acute ischemic stroke. Treatment with ancrod leads to fibrinogen depletion. The present study investigated the mechanisms leading to the reduction of plasma fibrinogen concentration. Twelve healthy volunteers received an intravenous infusion of 0.17 U/kg body weight of ancrod for 6 hours. Blood samples were drawn and analyzed before and at various time points until 72 hours after start of infusion. Ancrod releases fibrinopeptide A from fibrinogen, leading to the formation of desAA-fibrin monomer. In addition, a considerable proportion of desA-profibrin is formed. Production of desA-profibrin is highest at low concentrations of ancrod, whereas desA-profibrin is rapidly converted to desAA-fibrin at higher concentrations of ancrod. Both desA-profibrin and desAA-fibrin monomers form fibrin complexes. A certain proportion of complexes carries exposed fibrin polymerization sites E(A), indicating that the terminal component of the protofibril is a desAA-fibrin monomer unit. Soluble fibrin complexes potentiate tissue-type plasminogen activator-induced plasminogen activation. Significant amounts of plasmin are formed when soluble fibrin in plasma reaches a threshold concentration, leading to the proteolytic degradation of fibrinogen and fibrin. In the present setting, high concentrations of soluble fibrin are detected after 1 hour of ancrod infusion, whereas a rise in fibrinogen and fibrin degradation products, and plasmin-alpha(2)-plasmin inhibitor complex levels is first detected after 2 hours of ancrod infusion. Ancrod treatment also results in the appearance of cross-inked fibrin degradation product D-dimer in plasma. (Blood. 2000;96:2793-2802)     

SDS Polyacrylamide Gel Characterization of Serum FDP Produced in Response to Ancrod and Streptokinase/Plasminogen Infusions in Man

S ummary . The serum FDP produced in response to defibrination with ancrod and to thrombolytic therapy with intermittent streptokinase/plasminogen infusion have been characterized using a method of solid phase immuno-precipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Using this method it is possible to distinguish between the plasmin degradation products of fibrinogen and factor XIIIa induced crosslinked fibrin. During ancrod administration, fragments of similar mobility to fibrinogen fragments X, Y, D and E are present in serum samples taken 24 h after the initiation of treatment, while subsequent samples contain mainly fragments with the same mobility as fibrinogen fiagment D. The intermittent nature of the streptokinase/plasminogen infusions produces fibrinogen fragments X, Y, D and E in the serum 1 h after each daily streptokinase/plasminogen administration. An early clearance of fragment E from the circulation of patients receiving either ancrod or streptokinase/plasminogen infusions accords with the results of other investigations. The presence of the D dimer fragment, which is produced only by plasmin lysis of factor XIIIa crosslinked fibrin, could not be conclusively confirmed in either group of patients.     

Coagulation disturbances in cancer of the breast and colon measured with specific monoclonal antibody enzyme immunoassay for fibrin-fibrinogen degradation products

Disturbances of coagulation were measured in breast cancer, colon cancer and benign breast disease using an enzyme immunoassay against total plasma fibrinogen and fibrin degradation products (TDP). Results were compared with measurements of fibrinopeptide A (FPA) and of serum fibrinogen-related antigen (FRA). Plasma TDP before surgery was significantly higher in colon cancer than in breast cancer patients (p = 0.0009) or in benign breast disease (p = 0.0292). A post-operative rise in TDP at 4 months (colon cancer) or 9 months (breast cancer) was associated with early recurrent disease (p less than 0.0173 for colon cancer, p less than 0.1835 for breast cancer). No such association was noted with FPA or FRA measurements. TDP may prove to be of value in detecting activation of coagulation by malignant disease.     

Fibrinolysis and fibrinogenolysis in liver disease

Patients with liver disease frequently have hemostatic abnormalities which include accelerated fibrinolysis. In order to assess the fibrinolytic state in liver disease, plasma levels of fibrinogenolysis products (FgDP), fibrinolysis products (FbDP), and fibrinogenolysis plus fibrinolysis products (TDP) were measured with newly developed enzyme-linked immunosorbent assays based on monoclonal antibodies in 36 patients with liver disease (six patients with acute hepatitis, seven with chronic hepatitis, ten with liver cirrhosis, 11 with hepatocellular carcinoma, and two with intrahepatic cholestasis). As compared with healthy subjects, mean plasma levels of FbDP (1,083 +/- SD 1,254 vs. 236 +/- 100 ng/ml, P = 0.005) and TDP (1,773 +/- 1,814 vs. 669 +/- 212 ng/ml, P = 0.001) were significantly elevated in patients with liver disease, whereas FgDP was normal (389 +/- 202 vs. 396 +/- 132 ng/ml, P = 0.87). Plasma FbDP correlated very well with TDP (r = 0.986, P less than 0.00001) in liver disease. In addition, FbDP and TDP but not FgDP correlated with plasma concentrations of thrombin-antithrombin III complex. When plotted by the disease categories, the magnitude of elevations of FbDP and TDP was the most prominent in acute hepatitis followed by hepatocellular carcinoma. These findings indicate that activation of fibrinolysis occurs following thrombin generation, but increased primary fibrinogenolysis is rare in liver disease.     

Fibrin and fibrinogen degradation products with an intact D-domain C- terminal gamma chain inhibit an early step in accessory cell-dependent lymphocyte mitogenesis

Although the low molecular weight degradation products of fibrinogen (FgDP) and fibrin (FbDP) are known to inhibit lymphocyte blastogenesis, the effect of purified macro-molecular FgDP and FbDP (molecular weight, 90 to 200 Kd) is unclear. We have examined the effect of these latter FgDP and FbDP and find that products that contain the D domain inhibit lymphocyte proliferation in response to T-cell mitogens, allogeneic mononuclear leukocytes, and anti-CD3 in vitro. Plasmic digestion of D1 in the absence of calcium with removal of the C-terminal end of the gamma chain or disruption of the gamma-gamma C-terminal cross-link site of D-dimer (DD) by puffadder venom (PAV-D) abrogates their inhibitory potential. Prior incubation of monocytes with DD or D1 inhibits subsequent lymphocyte transformation. Binding studies with radiolabeled DD and PAV-D confirm that monocytes interact only with DD. This specific binding may be competitively inhibited by monoclonal antibodies to CD11b/CD18 or by peptide analogues of the C-terminal gamma chain of fibrinogen that mimic the adhesion recognition site of integrins. We postulate that DD and D1 bind to CD11b/CD18 on adherent monocytes and modulate lymphocyte activation. These products are typically present in the plasma of patients with disseminated intravascular coagulation with sepsis and could therefore influence inflammatory processes in vivo.     

Validity of enzyme-linked immunosorbent assays of cross-linked fibrin degradation products as a measure of clot lysis

Concentrations of cross-linked fibrin degradation products (XL-FDPs) in plasma, measured by enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) raised against fragment D-dimer of cross-linked fibrin, increase when patients are given fibrinolytic agents. Whether XL-FDPs derive from circulating cross-linked fibrin polymers in plasma, compared with clot-associated fibrin, has been questioned because increases in XL-FDP are measured by some assays after fibrinolysis in vitro in the absence of clot. We characterized the source of XL-FDP immunoreactivity in plasma of patients with acute myocardial infarction and ischemic heart disease and the response to plasminogen activation in vitro induced by pharmacological concentrations of tissue-type plasminogen activator (t-PA) and streptokinase. XL-FDPs were measured with two different ELISA. One, "pan-specific tag ELISA," was based on a capture MAb specific for XL-FDP and a tag MAb that recognizes an epitope exposed in the fragment D region of both fibrin and fibrinogen, whereas the other, "fibrin-specific tag ELISA," was based on a capture and tag MAbs both specific for fibrin. After plasminogen activation was induced in vitro in plasma from patients with myocardial infarction, increased concentrations of XL-FDP were measured by the pan-specific tag ELISA; however, concentrations measured with the fibrin-specific tag ELISA were not increased. To determine the mechanism for this discrepancy, plasma was subjected to immunoadsorption with a MAb specific for fragment D-dimer before and after in vitro activation of the fibrinolytic system and immunoblotting with a fragment D-dimer-specific MAb and with the pan-specific MAb. Increased concentrations of fragment D-dimer, as well as fibrinogen fragment D at high concentrations, were recognized by the specific MAb. Non-cross-linked fragments were also shown by immunoblotting with the pan-specific MAb to coprecipitate with cross-linked fibrin fragments. This suggested the increased concentrations of XL-FDP measured by the pan-specific tag ELISA after in vitro activation of the fibrinolytic system were due to detection of non-cross-linked fibrinogen fragments. However, fibrin fragment D-dimer concentrations were found to increase in plasma of 15 patients given t-PA for acute myocardial infarction. We conclude fragment D-dimer in plasma of patients during thrombolysis does not originate from circulating soluble cross-linked fibrin but rather is a marker of solid-phase fibrin dissolution, which may be quantitated with assays based on capture and tag antibodies that do not detect fibrinogen or its degradation products.     

Hemostatic imbalance in active and quiescent ulcerative colitis

OBJECTIVE: In healthy conditions, factors inducing or inhibiting coagulation and factors inducing or inhibiting fibrinolysis are in balance. In ulcerative colitis, hypercoagulation is presumed, which may explain part of the clinical features of this disease. Therapy strategies affecting hemostasis may improve the course of ulcerative colitis. This study was conducted to evaluate the balance of coagulation and fibrinolysis in the course of treatment of active ulcerative colitis. METHODS: Patients with active ulcerative colitis were studied by serial determination of markers of the coagulation cascade (thrombin-antithrombin complexes and fibrin degradation products [FbDP]) and the fibrinolytic cascade (fibrinogen degradation products [FgDP]). Parameters of inflammation were also measured (C-reactive protein [CRP], erythrocyte sedimentation rate [ESR], albumin, platelet count, and fibrinogen). Disease activity was assessed by endoscopic and histopathological scores. Follow-up measurement was performed in the course of treatment at the third or fourth month after baseline. Measurements were compared with healthy controls. RESULTS: Thirty-three patients and 22 healthy controls were included. During active ulcerative colitis, inflammatory parameters (CRP, ESR, platelet count) and hemostatic parameters (thrombin-antithrombin complexes, fibrinogen, FgDP, and FbDP) were elevated in comparison with healthy controls. Albumin was decreased and antithrombin-III remained unchanged. During treatment, disease activity decreased significantly endoscopically and histopathologically (p < 0.001). CRP, ESR, platelet count, and fibrinogen also decreased significantly. The hemostatic balance, expressed as the ratio between the plasmin-dependent generation of FgDP and coagulation-dependent generation of FbDP, increased from 0.69 to 1.12 during treatment, mainly because of a decrease of FbDP. In healthy controls, this ratio was CONCLUSIONS: The coagulation and fibrinolytic cascades were activated in active ulcerative colitis, with a hemostatic imbalance in favor of coagulation. This hypercoagulability persisted in 20% (7/33) of patients with ulcerative colitis in remission. The decrease of FbDP and the increase in the FgDP/FbDP ratio during reconvalescence of ulcerative colitis showed that the coagulation cascade was more activated than the fibrinolytic cascade in active disease.     

Fibrinopeptide A levels indicative of pulmonary vascular thrombosis in patients with primary pulmonary hypertension

Although the mechanisms involved in the pathophysiology of primary pulmonary hypertension have not yet been delineated, thrombosis has been implicated. This study was designed to determine whether thrombin activity as reflected by plasma concentrations of fibrinopeptide A (FPA), a marker of the action of thrombin on fibrinogen, is increased in patients with primary pulmonary hypertension. To evaluate fibrinolytic activity, we measured plasma concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cross-linked fibrin degradation products. We studied 31 patients with primary pulmonary hypertension. Plasma FPA concentrations measured by radioimmunoassay, were elevated to 87.4 +/- 36.9 ng/ml (mean +/- SEM). Fifteen minutes after administration of heparin (5,000 U), FPA concentrations decreased to 6.8 +/- 1.4 ng/ml (p less than 0.001 compared with preheparin levels). In 21 of 30 patients (70%), FPA concentrations after heparin administration were less than half the preheparin levels, a response consistent with inhibition of thrombin by heparin and the short half-life of FPA. Despite evidence for marked thrombin activity, plasma concentrations of cross-linked fibrin degradation products were normal in all but four patients. Plasminogen activator inhibitor-1 activity was elevated in 19 of the 27 patients in whom it was measured, potentially limiting the fibrinolytic response. The elevations of FPA indicate that thrombin activity is increased in vivo in patients with primary pulmonary hypertension. Thus, sequential assays of plasma markers of thrombosis and fibrinolysis in vivo may help identify those patients who may benefit from treatment with anticoagulants.     

Fibrinogen and fibrin degradation products in patients undergoing open-heart surgery

We have determined fibrinogen and fibrin degradation products (FgDP and FbDP respectively) by an ELISA method using specific monoclonal antibodies in 100 patients undergoing cardiopulmonary bypass (CPB) for valvular heart disease and in 60 patients undergoing aorto-coronary bypass surgery. Blood samples were taken pre-operatively and on post-operative days 1 and 5. Post-operative evolution was similar in both patient groups, with a significant increase in FgDP on post-operative days 1 and 5 with respect to baseline value (P less than 0.01). FbDP were also significantly higher on post-operative days 1 and 5 (P less than 0.001), especially the day after surgery in patients with valvular disease as compared with coronary patients (P less than 0.01). Our results indicate that fibrinolysis is more important than fibrinogenolysis after open-heart surgery, which may have pathophysiological implications.     

Inactivation of single-chain urokinase (pro-urokinase) by thrombin and thrombin-like enzymes: relevance of the findings to the interpretation of fibrin-binding experiments

Whereas crude bovine thrombin activated single-chain urokinase-type plasminogen activator (scu-PA), otherwise called pro-urokinase (pro- UK), purified human thrombin converted pro-UK (scu-PA) to a two-chain form that had no amidolytic activity. The two chains (Mr approximately 33,000 and 22,000) were disulfide linked and resistant to subsequent activation by plasmin. By contrast, thrombin did not inactivate tissue plasminogen activator or two-chain urokinase. The enzyme from snake venom Agkistrodon contortrix, relatively specific for fibrinopeptide B, had an effect similar to thrombin, whereas the enzyme from Agkistrodon rhodostoma (ancrod), specific for fibrinopeptide A, did not. When pro- UK (scu-PA) was present during thrombin clotting of fibrinogen, degradation of 125I-pro-UK (scu-PA) in the clot supernatant was seen, whereas virtually full recovery (95%) of radioactivity was found. A loss of latent amidolytic activity in the clot supernatant was also found, the extent of which could be correlated with the degree of degradation of the radiolabeled probe. It was concluded that thrombin inactivation of pro-UK (scu-PA) accounts for the loss of amidolytic activity in the clot supernatant, which has been attributed to fibrin binding. Further confirmation was obtained from experiments in which ancrod was used as the clotting agent. Full recovery of both radioactivity and latent amidolytic activity of pro-UK (scu-PA) in the supernatant was obtained under these conditions. These findings indicate that thrombin may introduce an artifact in the results of certain experiments designed to study the fibrin affinity or fibrinolytic effect of pro-UK (scu-PA).     

Characterization in vivo of the fibrin specificity of activators of the fibrinolytic system

Development of appropriate clinical dose regimens of individual plasminogen activators such as tissue-type plasminogen activator (t-PA) has generally relied primarily on nonpharmacological endpoints such as angiographically documented clot lysis. The recent availability of monoclonal antibodies that differentiate products of plasmin lysis of fibrin from those of lysis of fibrinogen should permit delineation of the relative fibrin specificity of different plasminogen activators or of different doses of the same activator in vivo. Thus, their use should accelerate and facilitate development of implementation of optimal dose regimens for diverse activators and combinations of activators. The present study was designed to determine whether assay of such markers effectively differentiates effects of two doses of t-PA, each of which are comparably effective in opening infarct-related arteries, in patients studied at the Washington University Clinical Unit of the National Institutes of Health-sponsored Thrombolysis in Myocardial Infarction Trial. The extent of lysis of fibrin and of lysis of fibrinogen by plasmin resulting from administration of t-PA was evaluated in 19 patients given 150 mg t-PA over 6 hours and 17 given 100 mg over the same interval by assay of serially obtained plasma samples for crosslinked fibrin degradation products (XL-FDP) and B beta 1-42, a peptide released when fibrinogen is degraded to fragment X by plasmin. XL-FDP were markedly elevated after 6-hour infusions of both doses of t-PA. However, elevations were not more with the higher dose [peak value, 4,321 +/- 986 ng/ml (+/- SEM)] compared with the lower dose (3,397 +/- 1,096 ng/ml) (p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interference of Lysed Fibrin with Coagulation: a Quantifying Approach Using Isotopic Techniques

S ummary . Coagulation of plasma fibrinogen in the presence of fibrin degradation products was studied in rabbits treated with ancrod (Arvin), Two techniques were employed: the first, based on trace-labelling of plasma samples with labelled fibrinogen in vitro , was to measure the degree to which solubilized fibrin inhibited coagulation; in the second, two differently labelled fibrinogens were used, one injected intravenously and the other added in vitro , by which estimation of fibrinogen in serum relative to the amount of circulating breakdown products was attempted.
The first technique is considered to provide reliable information about the extent of defective coagulation, and hence the proportion of masked fibrinogen present in serum. Because of the difficulty in obtaining resolution between fibrinogen and its breakdown products in clots, the second technique is thought to be more suitable for comparative studies rather than for measurements in absolute terms.
High doses of hydrocortisone administered to rabbits for several days prior to ancrod significantly reduced the speed of the dissolution of microclots. The data obtained are consistent with the hypothesis that the hormone interfered with fibrinolysis at the plasminogen activator level.     

经皮冠状动脉腔内成形术前后凝血功能的动态变化

目的了解经皮冠状动脉腔内成形术(PTCA)前后凝血及纤溶系统的相应变化.方法选择行PTCA的患者20例,于手术前1天、术后4、12、24、48、72小时和手术后第5天取血测定纤维蛋白肽A(FPA)、组织型纤溶酶原激活物(tPA)活性、纤溶酶原激活物抑制剂-1(PAI-1)活性.结果PTCA术后12小时FPA较术前增高(P＝0.01),并于术后24小时达到峰值(P＜0.01),均有非常显著性差异.此后逐渐降低,于术后第5天恢复至术前水平;PAI-1活性、tPA活性和PAI-1/tPA术前术后无明显变化.结论PTCA术后凝血系统被激活,应加强抗凝血酶的治疗.     

Factors relevant to stimulatory activity of fibrin degradation products in vivo.

Extracts of atherosclerotic lesions contain a range of fibrin degradation products (FbDP), similar fragments have been detected in extracts from human and mouse healing skin wounds and from the invasive edge of human breast carcinomas, which are all proliferating systems. We have previously shown that FbDP stimulate cell proliferation including angiogenesis in the chick chorioallantoic membrane (CAM), and sought to characterize further the active components. Fibrin prepared from platelet-rich and platelet-free plasma, and purified Kabi fibrinogen, was treated with plasmin, and the digests were all active. FbDP from platelet-rich plasma clots also increased vascularity of the CAM. Prior removal of fibronectin from plasma by gelatin-Sepharose affinity chromatography did not affect proliferative activity. Current studies showed that long digests of fibrin, in which the only major band detectable is fibrin fragment E are active. Commercial fibrinogen derived fragment E, itself inactive on the CAM, becomes active after exposure to thrombin cleavage of fibrinopeptides. Recently fragment E has been isolated from shorter digests, by simple filtration through a Millipore 0.2 microns centrifuge filter. It displayed similar activity to the fragment E obtained from long digests. Fragment E in plaque extracts has been shown consistently to lack fibrinopeptide A indicating it is of fibrin origin.     

Reaction of plasmic degradation products of fibrinogen in the radioimmunoassay of human fibrinopeptide A.

A radioimmunoassay (RIA) technique has been devised for the measurement of human fibrinopeptide A (FPA). The system utilizes rabbit antiserum to native human FPA and a synthetic fibrinopeptide, with tyrosine substituted for phenylanine in amino acid position 8. The test detects native human FPA at a concentration of 0.1 ng/ml, but does not cross react with human fibrinopeptide B or with fibrinopeptides A from canine, porcine, or bovine fibrinogen. Fibrinogen and chemical or plasmic degradation products with 2 moled of FPA per mole react fully in this test system. This includes the large-molecular-weight intermediate fragments X and Y and the NH2-terminal disulfide knot, and indicates that this antibody recognizes and reacts with FPA in the presence of the contiguous peptide structures present in fibrinogen. Fragment E, which is derived from the NH-2-terminal portion of fibrinogen, loses most of its FPA content after its liberation from its precursor derivative and reacts to a lesser extent in the RIA than do fragments X and Y. This correlated with the recovery of FPA-positive material from ultrafilitrates of extensive but not partial plasmic digests of fibrinogen. Although FPA immunoreactivity liberated from fibrinogen does not necessarily reflect thrombin activity and/or fibrin formation, only extensive plasmic degradation yields peptide material which reacts in this RIA system. This should not be a serious limitation to the application of the RIA in the detection of venous thrombosis.