An Abnormal Inherited Fibrinogen (Fibrinogen Genova) with Delayed Fibrin Aggregation

A new autosomally inherited dysfibrinogenaemia was recognized in 3 members of an Italian family. No bleeding tendency or thrombotic disease in any of the affected members were demonstrated. Coagulation tests revealed prolonged prothrombin, thrombin and Reptilase times. Plasma fibrinogen levels were normal with immunologic method and slightly reduced with chrono-metric assay: the other blood coagulation factors were normal. In addition, cross-immuno-electrophoresis performed on patients' plasma was indistinguishable from the normal. Dysfibrinogenaemia was confirmed by studying the purified fibrinogen. The fibrin polymerization curve, measured spectrophotometrically, showed a lower slope than the normal. A delay in fibrin monomer aggregation was revealed when compared to the normal at an equal concentration. The release of fibrinopeptides was normal. SDS polyacrylamide gel electrophoresis, isoelectric focusing and cross-immunoelectrophoresis of purified fibrinogen were not able to demonstrate any structural abnormality. The fibrinogen was named fibrinogen Genova.     

Dysfibrinogenaemia Characterized by Abnormal Fibrin Monomer Polymerization and Normal Fibrinopeptide A Release

S ummary . Routine testing on plasma from a patient due to undergo a coronary artery bypass graft operation revealed a prolonged thrombin clotting time associated with a normal plasma fibrinogen level when this was determined by a method not dependent upon the rate of fibrin formation. Fibrinogen purified from the patient's plasma by precipitation with β-alanine also gave a prolonged thrombin time and this confirmed the presence of a dysfibrinogenaemia. Increasing calcium chloride concentration, addition of protamine sulphate and decreasing ionic strength all produced a partial correction of the clotting defect. Addition of normal plasma to patient's plasma failed to correct the prolonged thrombin clotting time and a pH dependence of the defect was also observed. Kinetic studies of fibrinopeptide release, using a specific radioimmunoassay, demonstrated no delay in the release of patient fibrinopeptide A. The functional defect was localized as an abnormality in the polymerization of fibrin monomers by studying fibrin monomers prepared and isolated from plasma and from purified fibrinogen solution. An electrophoretic examination of the patient's fibrinogen using both agarose and polyacrylamide gels failed to demonstrate any alteration in mobility or any structural defect associated with the polypeptide chains Aα, Bβ and γ. All seven of the living siblings of the propositus and also his daughter showed no abnormality in any clotting assay. However, because the propositus did not suffer from liver disease it has been assumed that the abnormality is genetic in origin.     

The Identification of Fibrinopeptide B as a Chemotactic Agent Derived from Human Fibrinogen

S ummary . Human foetuses ranging in gestational age from 8 to 20 weeks were obtained at hysterotomy for therapeutic termination of pregnancy. Factor VIII related antigen was detected immunologically by Laurell's technique using specific antisera produced in rabbits. It was present in all foetal sera tested. It was not present in amniotic fluid and foetal cerebro-spinal fluid. An inhibitor of procoagulant factor VIII was neutralized by foetal serum but not by amniotic fluid. The distribution of factor VIII or factor-VIII-related antigen in foetal tissues was studied histologically by an indirect immunofluorescent technique. Specific fluorescence was seen on the endothelium of blood vessels in all tissues examined but in other cells the results were less conclusive. In tissue culture studies synthesis of factor-VIII-related antigen by explants of foetal spleen was demonstrated by incorporation of [³⁵S]L-methionine into specific immunoprecipitate. The results indicate that factor-VIII-related antigen and possibly procoagulant factor VIII or its precursor are synthezised at an early stage of foetal development.     

Radioimmunoassay of Human Fibrinopeptide A

A radioimmunoassay capable of measuring 1 pmole of human fibrinopeptide A has been developed, and should prove useful to detect the release of this peptide from fibrinogen during the coagulation process. Antibodies to fibrinopeptide A were produced by injecting New Zealand white rabbits with a mixture of Freund's adjuvant and native fibrinopeptide coupled to human albumin. N-Tyrosyl fibrinopeptide A was synthesized by the solid-phase method, and was iodinated with (125)I by the Chloramine-T method. 48-73% of the radiolabeled peptide could be bound by the serum of a rabbit immunized with the fibrinopeptide-albumin preparation. Antibody-bound peptide was precipitated by dioxane and was thus separated from unbound peptide. The addition of excess native fibrinopeptide to the radiolabeled material prevented its binding to serum. Native fibrinopeptide A and synthetic fibrinopeptide A were identical in their ability to prevent binding, whereas fibrinogen was from 1/25,000 to 1/50,000 as effective on a weight basis. Plasma filtered through a membrane relatively impermeable to molecules larger than a molecular weight of 34,000 showed no fibrinopeptide reactivity, whereas a similar filtrate of serum gave quantitative recovery of fibrinopeptide reactivity.     

Fibrinopeptide A binds Gly-Pro-Arg-Pro.

The tetrapeptide Gly-Pro-Arg-Pro inhibits fibrinogen aggregation, probably by binding to the same sites used during initiation of fibrin formation. The Gly-Pro-Arg-Pro binding sites have not yet been identified. However, their possible sequence and locations have been predicted on the basis of the amino acid pairing hypothesis. One of these predicted sites is on fibrinopeptide A. We report here that nuclear magnetic resonance studies indicate that Gly-Pro-Arg-Pro binds to fibrinopeptide A with a binding constant, K, of ca. 10(4) per mol. We also report results of 19 related peptide combinations used as controls.     

Fibrinopeptide A levels in patients with acute ischaemic heart disease

Fibrinopeptide A (FPA) levels were determined in 40 consecutive patients admitted to the coronary care unit with a typical history of chest pain: in 24 of them a diagnosis of acute myocardial infarction (AMI) and in 16 a diagnosis of angina was made. Seven of the patients with AMI suffered from recurrent episodes of early post-infarctional angina. FPA levels were determined in each patient on admission and every 4 h for 48 h. On admission in patients with both angina and AMI the FPA levels were significantly higher than in normal controls; these levels were higher in patients with AMI than in those with angina, but this difference was not significant. In patients with angina the values decreased progressively after 12 h to baseline values, while in those with AMI the high levels of FPA persisted throughout the 48-hour observation period. In many instances, particularly after 24 h, the differences between the two groups were statistically significant. In patients with early post-infarctional angina the episode of angor was preceded by or corresponded to a new great elevation of FPA levels. These data suggest that the thrombin generation is higher and more prolonged in patients with AMI than in those with angina; the determination of FPA levels, which are an index of 'in vivo' thrombin generation, can be useful to follow the clinical course of ischaemic heart disease.     

The Association of Fibrinopeptide A with Cardiac Events Following Coronary Angioplasty

Rethrombosis may be a major determinant of poor outcome after angioplasty. Fibrinopeptide A, a marker of thrombin activity, was measured in 79 patients after coronary angioplasty to assess for association with subsequent cardiac events. Fibrinopeptide A levels were drawn postangioplasty on heparin and prior to discharge off heparin. Levels greater than 2.0 ng/mL were considered elevated. During 6-month follow-up, recurrence of stenosis occurred in 14 out of 51 patients (27%) with elevated levels, requiring repeat angioplasty or bypass surgery. One more patient showed angiographic evidence of recurrence (at least 30% increase in stenosis) not severe enough to require revascularization. Fibrinopeptide A was elevated in this patient. There were three late cardiac deaths and one acute myocardial infarction, all in patients with elevated fibrinopeptide A. An additional five patients had an abnormal stress test without subsequent cathe-terization to assess recurrences; four of them had elevated fibrinopeptide A. In total, there were 22 out of 51 patients (43%) with elevated fibrinopeptide A levels with adverse outcomes, as compared with 6 out of 28 (21%) with normal levels (P = 0.05). Thus, an elevated fibrinopeptide A early after angioplasty may be a marker for a group at greater risk for restenosis.     

Obstetric Defibrination Syndrome with Abnormal Thrombin-Fibrinogen Reaction and Immunologically Reactive Altered Fibrinogen in Serum

The clinical and laboratory features of a patient with the obstetric defibrination syndrome are described. The abnormal bleeding responded to fibrinogen therapy and epsilon-aminocaproic acid was not used. The patient's plasma contained little fibrinogen capable of clotting without protamine and it inhibited the thrombin clotting time of normal plasma. Increased fibrinolysis was not observed. Serum obtained from the plasma after addition of protamine and concentrated thrombin contained a substance which reacted with an anti-fibrin serum when tested by an immunoelectrophoretic technique. The results confirmed that ‘altered’ incoagulable fibrinogen determinants were present in the patient's blood which may have interfered with the thrombin-fibrinogen reaction. It is suggested that these changes may be due to intravascular coagulation and not only to fibrinogenolysis. Similar but less severe coagulation changes were observed in the infant's cord blood.     

Quantitative Studies on the Release of Platelet Fibrinogen by Thrombin

S ummary . The effects of varying concentrations of thrombin on the release of fibrinogen from washed human platelets have been examined. A concentration of thrombin which gave adequate release and recovery levels of fibrinogen was used to study the time dependence of release. The small amount of thrombin required and the rapidity of the reaction indicates that the transfer of fibrinogen from intracellular to extracellular phase is due to an active release process.     

A Syndrome of Factor VII Deficiency and Abnormal Platelet Release Reaction

A 15-year-old girl with severe factor VII deficiency and chronic arthropathy showed an excessively prolonged bleeding time. Further studies demonstrated low platelet adhesiveness and abnormal platelet aggregation with ADP, collagen and epinephrine. Release of ¹⁴C-serotonin was deficient after aggregation with ADP and epinephrine, but was normal with thrombin. Transfusion of plasma or prothrombin complex concentrate resulted in a partial or complete correction of the bleeding time, respectively, but had no effect on in vitro platelet function tests. Both parents and the only sister had factor VII activities of 42 % - 72 % and factor VII antigen levels of 45 % - 66 % of normal and may thus be heterozygotes with respect to factor VII deficiency. All three had normal bleeding times in spite of abnormal in vitro platelet functions. The observations are interpreted to mean that in this family with factor VII deficiency and abnormal platelet release reaction the platelet abnormality as such was not sufficiently severe to prolong the bleeding time unless the factor VII activity was also very low.     

Platelet and Fibrinogen Survival in Normal and Abnormal States of Coagulation

1. Platelet and fibrinogen survival have been studied by isotope taggingin normal and abnormal states of coagulation in humans and dogs.

2. By our technic, the normal curve of platelet survival is exponential. Evidence is given that such a curve is obtained because of random utilization ofthe platelets in the continuous process of coagulation.

3. In the postoperative subject and in the subject receiving epinephrine injections, platelet survival is shortened because the process of coagulation isspeeded up. In the hypocoagulable, the curve of platelet survival becomesmore nearly linear because random platelet destruction ceases.

4. When a massive thrombus forms, platelet survival is shortened. Evidenceis given that this may be due to escape of thrombin from the clot into thesystemic circulation—with a resultant hypercoagulable state.

5. Fibrinogen survival was not altered in those states in which increasedplatelet utilization occurred. This is explained by the theory that the alterationin the hemostatic mechanism had not penetrated to the fibrinogen-fibrin stage.

6. Hypercoagulability is defined as that state in which platelet utilization isaccelerated—whether or not thrombosis occurs.

Submitted on February 17, 1960 Accepted on December 9, 1960     

高血压病患者血浆纤维蛋白原、载脂蛋白和异常形态红细胞的变化及其与腔隙性脑梗塞的关系

目的　研究高血压病患者血浆纤维蛋白原及载脂蛋白及异常形态红细胞变化特点及其与腔隙性脑梗塞 (腔梗 )的关系。方法　选择正常对照组 40例、高血压病组 45例和高血压腔隙性脑梗塞组 (腔梗组 ) 48例患者 ,分别测定血浆纤维蛋白原、载脂蛋白A(apoA)、载脂蛋白B(apoB)和血脂水平 ,并用扫描电镜观察了红细胞形态。结果　 (1)腔梗组血浆纤维蛋白原显著高于对照组与高血压病组 (P <0 0 1)。 (2 )腔梗组载脂蛋白A显著低于对照组与高血压病组 (P<0 0 1) ;腔梗组载脂蛋白B及载脂蛋白B/A比值显著高于对照组 (P <0 0 1)。 (3 )腔梗组平均异常形态红细胞检出率及异常形态红细胞增高率和脂蛋白B/A比值 >1的检出率均明显高于对照组及高血压病组 (P <0 0 1)。结论　血浆纤维蛋白原增高参与高血压病及高血压腔隙性脑梗塞的发生和发展 ,并提示血浆纤维蛋白原增高的程度可能预示病情程度。载脂蛋白A减低、载脂蛋白B增高与血浆纤维蛋白原增高有相似趋势 ,并可能与红细胞形态异常有关     

高血压病患者血浆纤维蛋白原、载脂蛋白和异常形态红细胞的变化及其与腔隙性脑梗塞的关系

目的研究高血压病患者血浆纤维蛋白原及载脂蛋白及异常形态红细胞变化特点及其与腔隙性脑梗塞(腔梗)的关系.方法选择正常对照组40例、高血压病组45例和高血压腔隙性脑梗塞组(腔梗组)48例患者,分别测定血浆纤维蛋白原、载脂蛋白A(apoA)、载脂蛋白B(apoB)和血脂水平,并用扫描电镜观察了红细胞形态.结果 (1)腔梗组血浆纤维蛋白原显著高于对照组与高血压病组(P<0.01).(2)腔梗组载脂蛋白A显著低于对照组与高血压病组(P<0.01);腔梗组载脂蛋白B及载脂蛋白B/A比值显著高于对照组(P<0.01).(3)腔梗组平均异常形态红细胞检出率及异常形态红细胞增高率和脂蛋白B/A 比值>1的检出率均明显高于对照组及高血压病组(P< 0.01).结论血浆纤维蛋白原增高参与高血压病及高血压腔隙性脑梗塞的发生和发展,并提示血浆纤维蛋白原增高的程度可能预示病情程度.载脂蛋白A减低、载脂蛋白B增高与血浆纤维蛋白原增高有相似趋势,并可能与红细胞形态异常有关.     

Fibrinopeptide A: a marker of acute coronary thrombosis

To determine whether coronary thrombosis in vivo is reflected by elevations in levels of fibrinopeptide A (FPA) in plasma, we sequentially characterized plasma FPA levels associated with evolving infarction in patients admitted to the cardiac care unit early after the onset of symptoms, in patients with transmural infarction admitted later, and in patients with nontransmural infarction. Studies were also performed in patients in whom the diagnosis of infarction was suspected but subsequently excluded. FPA values were significantly higher in patients with transmural infarction (42.3 +/- 11.2 ng/ml [mean +/- SEM], n = 53) compared with those in patients with nontransmural infarction (4.8 +/- 1.6 ng/ml, n = 17) or with those in patients in whom infarction was subsequently excluded as a diagnosis (3.5 +/- 0.6 ng/ml, n = 17, p less than .01 for both). Elevations in FPA level were greatest in patients with transmural infarction from whom samples were obtained soon after the onset of symptoms. Thus, in 39 patients from samples were obtained within 10 hr after the onset of symptoms, FPA levels were significantly higher than in 14 patients from whom samples were obtained initially more than 10 hr after the onset of symptoms (55.5 +/- 14.7 vs 4.9 +/- 1.4 ng/ml, p less than .01). In 30 of the 39 patients with evolving transmural infarction from whom samples were obtained within the first 10 hr after the onset of symptoms, the level of FPA was greater than 8 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinopeptide A and beta thromboglobulin in patients with angina pectoris and acute myocardial infarction

The purpose of this study was to investigate the degree of platelet activation and thrombin generation in 40 patients with stable angina pectoris and in 20 patients with acute myocardial infarction (AMI) by determining the plasma beta thromboglobulin (BTG) and fibrinopeptide A (FPA) concentrations. In patients with angina pectoris increased platelet activation correlated with extensive coronary pathology; the activation, however, was not influenced by a previous myocardial infarction, use of oral anticoagulants, beta-blocking agents, or hyperlipidemia. The plasma beta thromboglobulin concentration predicted more accurately the extent of the coronary artery disease than the functional angina pectoris classification. Thrombin generation was within the normal range. In patients with acute myocardial infarction increased platelet activation and enhanced thrombin generation were found, which were not related to the infarct localization, infarct size, or the presence of complications. Consequently, in these patients determination of plasma beta thromboglobulin and fibrinopeptide A concentrations is useless for the diagnosis of venous thromboembolism.     

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.     

Fibrinopeptide A and platelet factor levels in unstable angina pectoris

Fibrinopeptide A, platelet factor 4, beta-thromboglobulin, thromboxane B2, and 6-keto-prostaglandin F1 alpha were estimated by radioimmunoassay on venous plasma samples taken within 48 hr of admission from 16 consecutive patients with unstable angina and 15 patients with stable angina matched for clinical variables. The ratio of circulating platelet aggregates, platelet aggregation to increasing concentrations of ADP (0.455 to 1.82 micrograms/ml), and platelet thromboxane B2 production in vitro were also tested. The two groups of patients were statistically similar in terms of sex distribution, age, presence of risk factors, use of medication, extent of coronary artery disease and history of previous myocardial infarction. Mean plasma levels of fibrinopeptide A were 2.7 +/- 0.4 ng/ml (geometric means +/- SEM, range 1.5 to 5.5) in patients with stable angina vs 5.5 +/- 1.8 ng/ml (range 2.4 to 32; p less than .001) in those with unstable angina. In the latter group, after 6 to 8 days, fibrinopeptide A levels decreased to 3.6 +/- 0.5 ng/ml (range 1.5 to 9.3; p less than .04 vs admission). All other variables measured were statistically identical in the two groups. We conclude that plasma fibrinopeptide A levels, as opposed to platelet factors, discriminate between patients with unstable and stable angina, indicating an activation of the coagulation system in unstable angina.