Fibrin and fibrinogen proteolysis products: comparison between gel filtration and SDS polyacrylamide electrophoresis analysis.

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400,000 to 800,000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and nonstabilized fibrin in that fragment D was present in the "double D" cross-linked form.

Reduction of coagulation factor XIII concentration in patients with myocardial infarction, cerebral infarction, and other thromboembolic disorders.

Coagulation factor XIII and plasma fibrinogen chromatographic assays have been performed serially in patients suffering from acute myocardial and cerebral infarction, and in others with disseminated intravascular coagulation. The findings were compared with 2 groups of "controls"; normal clinically-well subjects and hospitalized patients with cerebral infarction who exhibited minimal, stable, or improving neurological deficits. Substantial depression of factor XIII concentrations developed in the 3 patient groups, together with concomitant significant increases in the proportion and concentration of plasma high molecular weight fibrin(ogen) complexes (HMWFC). An inverse correlation (p less than 0.05) between coagulation factor XIII concentration and percentage of HMWFC was demonstrated in the early stages of the illness. These findings suggest that depression of coagulation factor XIII concentration in these states, is secondary to extravascular or intravascular coagulation and may reflect its degree.

Association between oral contraceptive use and thromboembolism: a new approach to itsinvestigation based on plasma fibrinogen chromatography.

Longitudunal and cross-dectional blood coagulation studies were made in patients receiving oral contraceptive therapy and in unmedicated women used as control subjects. These studies have included use of a new procedure, plasma fibrinogen chromatgraphy, which, by assay for a high molecular weight fibrinogen complexes in plasma, detects or excludes the presence of small thrombi, even when these are of the clinically silent type. New contraceptives users (n=154) received either Ovulen (100 pg of mestranol) or Demulen (50pg of ethinyl estradiol) and were followed serially for one year. During the cross-sectional study (193 women and 1,350 samples), serial examination was preformed on those taking oral contraceptives for 3 months to 10 years. Over-all, pathologic plasma fibrinogen chromatographic findings, indicative of thrombosis, were detected in 6 per cent of hte control examinations and in 27 per cent samples from oral contraceptive users. These findings suggest that oral contraceptive users developed mainlyclinically silent thrombotic lesions, with four-to-fivefold greater frequency than the control subjects. Consequently, it is inferred that they are at four-to fivefold greaterrisk of developing clinically overt disease, a risk factor estimate in line with that derived by epidemiologic study.

Formation of soluble fibrin oligomers in purified systems and in plasma.

The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, the conditions of the present study, Vmax. was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation in purified fibrinogen incubated with thrombin proceeded as a bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of calcium were similar in composition to those without, i.e. 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-Mr variety (tetramer and greater); these high-Mr polymers contained less than 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e. 2 mol of FPA/mol.

Coagulation factor XIII concentration in sickle-cell disease.

Plasma factor XIII was assayed serially for a 1 1/2 to 2 year period in four patients with sickle-cell disease (SSD), during which several episodes of SSD crisis were observed. During crisis-free periods, plasma factor XIII concentration was lower than in normal subjects. With crisis onset, there was usually a concomitant fall in platelets and in plasma factor XIII soon after followed by a rise in fibrinogen concentration and platelet count. Following crisis resolution there was an increase in plasma factor XIII concentration peaking 3 weeks after crisis onset and occurring at the time of subsidence of thrombocytosis. It is suggested that the observed changes in plasma factor XIII concentration in SSD patients support the hypothesis that in the early stage of SSD crisis, fibrin deposition and/or thrombosis significantly enhance the role of sickled erythrocytes in producing vascular occlusion and/or organ infarction.

Fibrinogen catabolism in patients with type II and type IV hyperlipidemia. Effect of dietary and clofibrate treatment on laboratory findings.

Fibrinogen catabolism was studied by plasma fibrinogen chromatography and other methods in 99 subjects with hyperlipoproteinemia Types IIa, IIb, and IV, and in 24 control subjects with normal blood lipid values. Subjects with either a history of thromboembolic vascular disease or clinical evidence of atherosclerosis were excluded. Type II subjects (i.e., the combined group of Type IIa and IIb subjects) showed an elevation of plasma high molecular weight fibrinogen complexes, which is indicative of enhanced fibrin formation. They also showed an elevation of fibrinogen-first-derivative, which is indicative of fibrinogenolysis and increased plasma euglobulin activity. Subjects with Type IV hyperlipoproteinemia showed similar findings to those of Type II except that high molecular weight fibrinogen complex concentration was normal. Subsequently, 36 patients received a fat-controlled, low cholesterol diet and were studied in a blind, random, crossover study of dietary vs clofibrate treatment. Although total cholesterol and triglyceride levels fell significantly during the treatment periods, chromatographic findings of abnormal plasma fibrinogen remained unchanged.

Hemostatic alterations accompanying sickle cell pain crises.

Three patients with sickle-cell disease (SSD) were followed, weekly, for 1 1/2 to 2 years, during which time they experienced one or more episodes of crisis. Crisis was associated with reproducible sequential hemostatic alterations indicating intravascular fibrin formation and a marked disturbance in platelet economy. With crisis onset, or possibly before, there was an increase in plasma high-molecular-weight fibrinogen complexes and a transient fall in platelet count, with a subsequent rise in both fibrinogen concentration and platelet count; plasma fibrinogen peaked 1 week after crisis onset and platelet count approximately 2 weeks after onset. Subsidence of crisis was associated with a fall in high-molecular-weight fibrinogen complexes and a subsequent increase in fibrinogen first derivative, an early fibrinogen breakdown product. Hemostatic findings and patient clinical status were generally correlated, the findings during asymptomatic periods being essentially normal. Agents affecting platelet function (aspirin alone or in combination with dipyridamole) appeared to reduce the extent of laboratory abnormality, suggesting potential clinical usefulness in this disorder.

Course and resolution of the coagulopathy in nephrotic children.

Blood coagulation function was serially studied in 84 children with nephrotic syndrome. Fifty-eight had minimal change disease, six had focal glomerulosclerosis and 20 had other forms of renal disease associated with the nephrotic syndrome. Qualitatively similar abnormalities in fibrinogen metabolism were present in all groups with clinically overt nephrotic syndrome; plasma fibrinogen concentration and high molecular weight fibrin(ogen) complexes (HMWFC) were grossly elevated (P less than 0.001 in most groups). With disease remission fibrinogen and HMWFC concentrations decreased to the normal range, usually with concomitant transient increase in plasma fibrinolytic activity (P less than 0.02). Alterations in concentrations of other proteins involved in coagulation and fibrinolysis differed depending on the underlying cause for the nephrotic syndrome. Antithrombin III concentration was normal except in the focal glomerulosclerosis group. The results demonstrate that a coagulopathy characterized by pathological degree of thrombin action on fibrinogen complicates the nephrotic state and may be initiated by different mechanisms. It is suggested that this coagulopathy, which remits with clinical improvement, is consequent upon local intrarenal activation of the blood coagulation system.

Blood coagulation system pathophysiology in acute myocardial infarction: the influence of anticoagulant treatment on laboratory findings.

Two hundred twenty patients admitted to a Coronary Care Unit were studied by serial plasma fibrinogen chromatography--a method for quantification of HMWFCs, native fibrinogen, and other fibrinogen derivatives in plasma. Enhanced formation of fibrin (intravascular coagulation/thrombosis) is reflected by elevation of plasma HMWFC. One hundred ten patients suffering from documented acute myocardial infarction showed early, sharp elevation of plasma HMWFC (p less than 0.001 when compared to normal and cardiac control groups), a finding which persisted for 10 to 20 days after infarction. Forty-three of the patients did not receive anticoagulant therapy, and the others received either initial heparin, heparin plus warfarin, or werfarin therapy. Plasma fibrinogen chromatographic findings, days 1 to 5, did not differ between the anticoagulated and nonanticoagulated treatment groups, although there were minor differences in the data for days 6 to 10. The results demonstrate that patients with acute myocardial infarction develop a coagulopathy characterized by enhanced fibrin formation, which is influenced to only a minor degree by conventional dosage anticoagulant therapy.

Fibrinogen catabolism in burned patients.

Plasma fibrinogen chromatography and other relevant determinations of factors of known importance in thrombus production and dissolution were done serially in 19 hospitalized burned patients. The findings indicate that the coagulation mechanism was chronically activated and that, judging from the observed, sustained elevation of circulating high molecular weight fibrin (ogen) complexes, intravascular clotting was occurring at a pathological, if variable, rate. In patients older than 50 years of age, a relatively impaired thrombolytic response was also found; the latter finding is of special interest, as it provides biochemical substantiation for the well-recognized clinical proclivity of elderly burn patients to thromboembolic complications.

Blood coagulation in postmenopausal women given estrogen treatment: comparison of transdermal and oral administration.

The responses of the blood coagulation and related systems were studied in 23 postmenopausal women, all of whom received, in randomized order, therapy with conjugated oral estrogens (0.625 and 1.25 mg daily) and transdermally administered estradiol in doses of 25, 50, 100, and 200 micrograms/24 hr. Neither plasma fibrinopeptide A determinations nor plasma fibrinogen chromatographic findings were altered; thus there is no evidence of accelerated fibrinogen turnover with either form of therapy. However, alpha 1-antitrypsin and plasminogen concentrations were significantly increased with the higher dosage of oral but not with transdermally administered estrogen. Similarly, ceruloplasmin concentration was significantly elevated with both oral doses but was unchanged by transdermal therapy.

Severe glomerulonephritis complicated by coagulopathy: treatment with anticoaguland and immunosuppresive drugs.

Serial determinations, using plasma fibrinogen gel chromatography as well as standard methodology, demonstrated that six children with severe glomerulonephritis, characterized on renal biopsy by glomerular necrosis and crescent formation, had persistent evidence of intravascular coagulation. Based on these observations, therapy with anticoagulants and azathicoagulants and azathioprine was instituted for one year; treatment with anticoagulants was continued for a second year. Anticoagulant therapy was initiated with heparin, followed by oral anticoagulation with phenindione and dipyridamole. In contrast to our earlier experience with similar patients, each of the present patients improved. Urinalyses returned to normal and glomerular filtration rates to near normal values in all patients at the end of the treatment period and have remained so for up to 3.9 years since treatment has been completed. Post-treatment biopsies showed remarkable improvement, with virtually no glomerulosclerosis even in patients who had had a high incidence of glomerular crescents before treatment. It is suggested that the therapeutic regimen favorably influenced the natural history of disease and that plasma fibrinogen chromatographic findings may be helpful in selecting patients likely to benefit from the use of anticoagulant therapy.

Blood changes in atherosclerosis and long after myocardial infarction and venous thrombosis.

The following clinical groups of volunteers were studied: patients long after recovery from myocardial infarction (MI), others after recovery from deep vein thrombosis (DVT), patients with intermittent claudication, with diabetes, and male and female controls who were well matched. All were subjected to many platelet and clotting tests together with clinical, biochemical and haematological measurements in an attempt to find long term abnormalities in these various diseases. The male MIs differed very significantly from the controls in having much more heparin neutralizing activity (P less than 0.001)and less anti-thrombin (P less than 0.01). Less significantly, some bleeding time tests indicated less bleeding and the patients' platelets were larger. The females with MI had in general the same abnormalities but to a lesser degree. The patients with intermittent claudication, none of whom had a history of MI, had almost the same abnormalities and to the same degree. In deep vein thrombosis the heparin neutralizing activity was also clearly increased; the other tests were generally in the same direction but many were not significant. The diabetics had shorter bleeding times but little else abnormal relative to the controls, suggesting a different pathological process. When all male patients and controls were "scored" according to the degree of atherosclerosis there was a close overall correlation between the degree of atherosclerosis and the increase in the HNA level (r = --0.50, n = 66, P less than 0.001) and the decreased anti-thrombin (r = 0.25, n = 66, P less than 0.05).

Blood coagulation and plasma fibrinolytic enzyme system pathophysiology in stroke.

Plasma fibrinogen chromatography is a method for quantification of high molecular weight fibrinogen complexes (HMWFC), native fibrinogen and other fibrinogen derivatives in plasma. Enchanced formation of fibrin, intravascular coagulation, thrombus formation, etc., are reflected by elevation of plasma HMWFC, and the method distinguishes between subjects with normal and pathological rates of fibrin formation. Serial standard blood coagulation assays, including plasma fibrinogen chromatography, and neurological studies were performed on 220 patients admitted to a stroke unit. Findings from patients with cerebral infarction were compared against those of three control groups: (1)normals, (2)a stroke control group and(3)a stroke risk factor group. Plasma HMWFC findings were significantly (p less than 0.001) higher in the stroke risk factor group than in the normals. Plasma HMWFC values were significantly higher (p less than 0.001) in the cerebral infarction patients than in any of the control groups, and plasma fibrinogen, plasminogen, alpha1-antitrypsin and alpha2-macroglobulin also were significiantly higher (p less than 0.001) in the patients. The greater the degree of initial neurological deficit, the greater were plasma HMWFC values (p less than 0.001), and high HMWFC values were associated with poor clinical outcome. Plasma HMWFC values were significantly higher (p less than 0.001) in patients with intracerebral hemorrhage, subarachnoid hemorrhage and cerebral embolism. These findings docunment the fact that a high proportion of stroke patients have coagulopathy, characterized by pathological enhancement of fibrin formation.

Pathophysiological response of the blood coagulation system in acute glomerulonephritis.

A new procedure, plasma fibrinogen chromatography, has been utilized, together with other blood coagulation assays, to quantify fibrin formation in 43 children with acute poststreptococcal glomerulonephritis (AGN) from the time of hospitalization until recovery. During the prediuretic phase of AGN, significant evidence for substantial increase in fibrin formation (intravascular coagulation) included gross increase in plasma high molecular weight fibrinogen complexes (HMWFC), the development of either hypo- or hyperfibrinogenemia and gross depression of coagulation factor XIII concentration and of alpha2-macroglobulin concentration. During the diuretic phase of the disease, these abnormalities regressed and evidence of enhanced plasma fibrinolytic activity, documented by an increase in fibrinogen first derivative, was detected. Concomitantly, urinary excretion of fibrin(ogen) degradation products (FDP) underwent substantial increase. With disease recovery, which occurred in all children, urinary FDP excretion ceased and all coagulation findings normalized.

A pilot study of urokinase therapy in cerebral infarction.

Thirty-one patients with acute cerebral infarction were treated with the thrombolytic agent urokinase for either a single or a double infusion period, each of ten hours. The effects of urokinase therapy administered at dosage rates of 1,200, 1,500 or 1,700 CTA urokinase units per pound of body weight per hour were followed by serial blood coagulation and other biochemical studies. In the dosage used, urokinase produced a prompt sustained increase, 20-fold to 40-fold, of plasma thrombolytic activity with relatively minor disturbance of the blood coagulation system. Nevertheless, hemorrhagic complications occurred in several patients and distinctly favorable therapeutic effects were not observed.