Prolonged Thrombin Time Research Articles

A deficiency in Aα chain's N-terminal alanine residue as a major cause of the slow coagulation of fetal fibrinogen

To clarify the mechanism which causes the coagulation time of fetal fibrinogen to be longer than that of adult fibrinogen, the N-terminal amino acid residues of fetal fibrinogen were analyzed. The results showed that the amount of Aa chain's N-terminal alanine residues in fetal fibrinogen was only 54% of that in adult fibrinogen. When the amount of Aα chain's N-terminal residual alanine in adult fibrinogen was decreased to 57% of the normal level by digestion with aminopeptidase M, the adult fibrinogen yielded a prolonged thrombin time and a retarded release rate for fibrino-peptides A and B, both values approximating to those of fetal fibrinogen. The results suggest that the deficiency in alanine residue at the N-terminus of the Aa chain is a major cause of the slow coagulation of fetal fibrinogen.

Disturbed fibrinolysis in patients with inflammatory bowel disease. A study in blood plasma, colon mucosa, and faeces.

Using specific assays, we studied fibrinolytic activity in plasma and colonic mucosa biopsies of 28 patients with inflammatory bowel disease (IBD) (12 with Crohn's disease, 16 with ulcerative colitis) and 28 control patients without inflammatory bowel disease or colon malignancy. Blood coagulation was studied using standard techniques. In plasma of IBD patients significantly decreased tissue type plasminogen activator activity (t-PA) (p less than 0.02), increased plasminogen activator inhibition (PAI) (p less than 0.01) and fibrinogen (p less than 0.001), and prolonged thrombin time (p less than 0.001) and prothrombintime (p less than 0.001) were found. In colon mucosa the percentage of t-PA to urokinase type plasminogen activator (u-PA) was 80:20% in the control group and 71:29% in the IBD group in non-inflamed mucosa. In inflamed mucosa the plasminogen activator percentage was 55:45%, significantly different (p less than 0.01) from the control group. There was also a significant absolute increase in u-PA activity and decrease of t-PA activity in the inflamed mucosa compared to the control group (p less than 0.001 and p less than 0.01, respectively). Patients with inflammatory bowel disease therefore have significant changes in components of the fibrinolytic and coagulation system both systemically and locally in colon mucosa. These changes might contribute to an increased risk for thromboembolic complications and possibly to the pathogenesis of the colitis and to the local complications such as bleeding.

The Polymerization of Fibrin Prepared from Fibrinogen Haifa (γ275Arg→His)

Fibrinogen Haifa is a congenital heterozygous fibrinogen variant (gamma 275 Arg----His) characterized by prolonged thrombin and reptilase times and normal fibrinopeptide (FPA, FPB) release. We compared the polymerization rate (by turbidity measurements at 350 nm) and the ultrastructure of Haifa alpha-, beta-, and alpha, beta-fibrin with that of normal. Haifa alpha, beta-fibrin polymerized less rapidly than did normal and formed a highly branched matrix with a smaller mean fiber diameter; this network closely resembled that of normal alpha, beta-fibrin with EDTA added. In the presence of CaCl2 (1 to 10 mM), Haifa alpha, beta-fibrin polymerized more rapidly than in buffer alone and possessed a matrix structure closely resembling that of normal fibrin. From these observations it appears that the functional defect in Haifa fibrin can be related to the inability of the abnormal molecule to effectively utilize available calcium. The polymerization profile of Haifa alpha-fibrin differed only modestly from that of normal alpha-fibrin, whereas that of Haifa beta-fibrin was markedly impaired. This finding plus similarities in the ultrastructure of Haifa and normal alpha-fibrin specimens suggests that the defective gamma chain structure of Haifa fibrinogen results in greater impairment of the carboxy terminal "b" polymerization domain reacting with the site exposed by cleavage of FPB ("B" site) than it does that of the carboxy terminal "a" domain reacting with the site exposed by cleavage of FPA ("A" site). Whether this effect is due to absolute differences in the degree of impairment of these two types of polymerization sites, or whether proper utilization of the "B" to "b" site is dependent upon participation of the "A" to "a" site remains to be determined.

Fibrinogen Baltimore III: Congenital dysfibrinogenemia with a shortened γ-subunit

An abnormal fibrinogen has been found in an asymptomatic Negro female. Clinical laboratory findings were normal, except for a prolonged thrombin time which was corrected by addition of calcium. Fibrinopeptide release by thrombin and crosslinking by factor XIII also occurred normally, but fibrin monomer polymerization was delayed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis disclosed that 50% of the γ-subunits migrated with an apparent M r of 45,500, approximately 1,500 Da smaller than normal. The evidence suggests that an internal sequence of 10–15 residues is missing from the γ-subunit of the abnormal fibrinogen.

Association between necropsy evidence of disseminated intravascular coagulation and coagulation variables before death in patients in intensive care units.

The necropsy findings in 21 patients on an intensive care unit, on whom coagulation studies had been performed immediately before death, were assessed. Eleven of the patients were retrospectively studied and 10 were reviewed consecutively in a prospective study. Fifteen patients (eight retrospective and seven prospective) had evidence of disseminated intravascular coagulation. Microthrombi were most often found in the lungs and kidneys. The most common abnormal coagulation tests in patients with necropsy evidence of disseminated intravascular coagulation were raised serum concentrations of fibrinogen and fibrin degradation products, prolonged prothrombin time, and reduced platelet counts. Reduced fibrinogen concentrations and a prolonged thrombin time were the least commonly observed abnormalities. There was no difference in either the prevalence or magnitude of abnormality of any particular coagulation variable test result between those patients with evidence of disseminated intravascular coagulation at necropsy and those without.

A Study of Fibrinogen and Fibrinolysis in 10 Adults with Nephrotic Syndrome

In 10 patients with nephrotic syndrome (NS), the coagulation inhibitors, the fibrinolytic system and several functions of the fibrinogen-fibrin molecule were studied. Among the coagulation inhibitors, only antithrombin III (AT III) was found decreased and correlated with serum-albumin levels. Venous occlusion test provoked a normal tissue plasminogen activator (tPA) release in all patients. The plasminogen activator inhibitor (PAI) had an increased activity in 5 out of the 10 patients. Thrombin and reptilase times were found abnormal in most patients. The thrombin time (TT) prolongation correlated with serum albumin levels and was corrected by adding purified albumin. The fibrinogen was purified from each of the 10 patients' plasma. Only 2 of them showed abnormal polymerization in purified system, suggesting dysfibrinogenaemia. Other functions (thrombin binding, tPA stimulating activity, lysis by purified plasmin) were found normal except in one of the 2 patients with dysfibrinogenaemia whose fibrinogen lysis by plasmin was delayed. It is concluded that an abnormal fibrinogen molecule is not the most frequent explanation for thrombin time prolongation in NS.

Characterization of Fibrinogen Milano I: Amino Acid Exchange γ330Asp → Val Impairs Fibrin Polymerization

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called “fibrinogen Milano l.” The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303–356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

Multiple Myeloma With Polyneuropathy and Coagulopathy

• A patient with the syndrome of polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) had multiple myeloma and IgA-κ monoclonal gammopathy. To our knowledge, of over 50 cases in the literature, this is the only instance of κ light chains noted in a patient with the complete syndrome. Sural nerve biopsy revealed increased immunofluorescence for IgA and κ chains. Furthermore, this patient had a hemorrhagic diathesis characterized by a prolonged bleeding time and a prolonged thrombin time due to a qualitative platelet dysfunction and an apparent inhibition of fibrin monomer polymerization. In none of the other cases of POEMS syndrome was a coagulopathy reported. A short course of plasmapheresis improved the bleeding time but did not alter the polyneuropathy. (Arch Intern Med1986;146:993-994)

Three Abnormal Fibrinogen Variants with the Same Amino Acid Substitution (γ 275 Arg → His): Fibrinogens Bergamo II, Essen and Perugia

We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and reptilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (gamma 265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified gamma-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: gamma 275 arginine----histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.

Heparin-Like Anticoagulant Associated with Plasma Cell Myeloma

A 54-year-old man with plasma cell myeloma had sustained bleeding develop after prophylactic hip hemiarthroplasty. Routine coagulation studies revealed significant prolongation of the prothrombin time, activated partial thromboplastin time, and thrombin time. Further evaluation showed failure of the activated partial thromboplastin time to correct in a 1:1 mixture with pooled normal plasma, correction of the prolonged thrombin time by addition of protamine, and a normal reptilase time. A purified preparation of the immunoglobulin component of patient plasma produced the same pattern of coagulation abnormalities, suggesting the paraprotein possessed heparin-like anticoagulant activity. This appears to be a rare mechanism of bleeding diathesis in plasma cell myeloma.

Unrecognized pulmonary embolism presenting as disseminated intravascular coagulation

Six patients are described in whom disseminated intravascular coagulation of uncertain cause was found to be due to occult pulmonary embolism. The peripheral blood smear showed thrombocytopenla in all patients and schistocytes in four. Coagulation studies revealed increased levels of fibrinogen/fibrin degradation products (six of six patients), positive results for fibrin monomer (five patients), prolonged thrombin times (four patients), hypofibrinogenemia (three patients), prolonged prothrombin times (two patients), and decreased plasma coagulation factors (two patients). Pulmonary embolism was confirmed by lung scanning or pulmonary angiography. Institution of full-dose heparin therapy was associated with hemostatic and clinical improvement in all patients. The association of disseminated intravascular coagulation with occult pulmonary embolism merits recognition since full-dose heparinization is required for successful therapy.

Granulocyte enzyme mediated degradation of human fibrinogen in plasma in vitro

Incubation of plasma with granulocyte enzymes in the presence of kallikrein inhibitor resulted in a prolonged thrombin time of the plasma and in an increased level of fibrinogen fragments, indicating that fibrinogen was digested. In accordance with this, fibrinogen digestion products could be purified by affinity chromatography from plasma after incubation with granulocyte enzymes. The isolated products resembled early X-like fibrinogen fragments, which are produced by limited digestion of purified fibrinogen with elastase. On SDS gel electrophoresis both had no intact Aα-chains, but apparently intact fibrinogen Bβ- and γ-chains. Also, both fragments isolated from plasma and the X-like fragments produced with purified elastase had a low anticoagulant activity. Although elastase, the main fibrinolytic enzyme of the granulocyte, was rapidly complexed with inhibitors, 10–20% of the elastase activity towards succinyl-trialanyl-paranitro-anilide was detectable in plasma no matter if the mixture of granulocyte enzymes or purified elastase had been added. A possible role for the α 2-macroglobulin-granulocyte elastase complex in the production of the digestion products in plasma is discussed.

Acquired haemostatic defects in the ill newborn.

Summary. The kinetics and function of platelets and fibrinogen were studied in 22 ill newborns. Overall 51Cr‐platelet survival was considerably shortened (patients 2·6 d ± 1·7, adult normal 9·5 d ± 0·6, P < 0·001) while 125I‐fibrinogen surival was less severely decreased (patients 2·1 d ± 0·8, adult normal 5·1 d ± 0·4, P < 0·05).Three patients with bacterial sepsis and one with disseminated herpes simplex had the most severely reduced platelet survival times (1·3 d ± 0·9), causing thrombocytopenia (62 ±32 × 109/l) despite production rates averaging 2 1/2 times normal. Although fibrinogen survival times were comparably reduced in these patients to 1·4 d ± 0·4, fibrinogen concentrations were normal because of compensatory increased production.In three patients with necrotizing enterocolitis platelet and fibrinogen survival times were similarly reduced (platelets 1·6 d ± 0·6, fibrinogen 1·6 d ± 0·6) with platelet counts of 69 ± 27 × 109/l and platelet production rates of twice normal. Since their fibrinogen production was not increased, they had decreased fibrinogen concentration (1·0 ± 0·19 g/l).In six patients with respiratory distress syndrome the kinetic results were more variable. Platelet survival was less severely affected than in the other groups and production was approximately twice normal; hence, the platelet count was maintained (177 ± 107 × 109/l). Fibrinogen in this group was slightly reduced because of decreased production in the presence of mildly decreased survival (2·7 d ± 0·7).The two patients with cytomegalovirus infection had severe thrombocytopenia (23 × 109/l) due to a combination of decreased platelet survival (3·6 d), increased splenic pooling and no compensatory increase in marrow platelet production. Fibrinogen production was normal but its plasma concentration was moderately reduced because of a decrease in fibrinogen survival.In many of these patients, fibrinogen dysfunction was suggested by a prolonged thrombin time and a discrepancy between fibrinogen concentration as determined by a time dependent assay and an equilibrium total clottable protein assay. Likewise, platelet dysfunction, evidenced as a prolonged bleeding time, was found in patients with RDS; some of these infants had otherwise unexplained bleeding.

Idiopathic autoantibody that inhibits fibrin monomer polymerization

A 73-year-old female was found to have prolonged thrombin and reptilase times in the immediate post-operative period. These abnormalities were not corrected by the addition of normal plasma. They were subsequently shown to be due to an IgG immunoglobulin which inhibited fibrin monomer polymerization. The IgG immunoglobulin activity could be neutralized completely by prior incubation with either patient or normal fibrinogen, uncrosslinked fibrin monomers or IgG antisera. No inhibitory effect on thrombin activity, fibrinopeptide A release or on the fibrin cross-linking reaction of factor XIIIa could be detected. Purified patient fibrinogen was functionally normal as demonstrated by normal fibrinogen-fibrin polymerization and fibrinopeptide A release. No underlying cause for this phenomenon was found. The presence of the inhibitor was associated with excessive blood loss during the post-operative period.

Bleeding and coagulation abnormalities in alcoholic cirrhotic liver disease.

Coagulation profiles were performed in 30 consecutive alcoholic cirrhotic patients without known infection, malignancy, recent surgery, transfusion, or alcoholic intake. Hemorrhagic phenomena were present in 70% and included gastrointestinal bleeding, oozing from venipuncture sites, bruising, and epistaxis. All 30 patients had multiple liver function and coagulation abnormalities, the most frequent of which were increases in F VIII components and decreases in F XI and F VII. Also decreased in half or more of the 30 patients were Fletcher F, F II, F X, prothrombin time (PT), partial thromboplastin time (APTT), thrombin time (TT), reptilase time (RT), anti-thrombin III, and plasminogen. When comparing cirrhotic bleeders with nonbleeders, four parameters were significantly different in those with a bleeding tendency: F VII, anti-thrombin III, plasminogen, and albumin. The prolonged APTT was associated in four cases with a blocking inhibitor of unknown etiology. The prolonged TT and RT, in the absence of fibrin split products, fibrin monomers, DIC, or shortened euglobulin lysis time in any patient were suggestive of an abnormal fibrinogen, a dysfibrinogen. In three other patients, there was an inhibitor of the TT. Further investigation of the suspected dysfibrinogen in 21 patients by SDS-polyacrylamide gel electrophoresis revealed that the molecular weights of the Aalpha, Bbeta, and gamma polypeptide chains of fibrinogen were not different from normal. Two-dimensional immunoelectrophoresis of the suspected dysfibrinogen was similar to normal in 18 of 21 patients, with loss of the initial shoulder in three.

Microangiopathic hemolytic anemia, thrombocytopenia, and renal failure in patients treated for adenocarcinoma.

Microangiopathic hemolytic anemia and thrombocytopenia secondary to disseminated intravascular coagulation is a well-described complication of widely metastatic carcinoma. The authors report four cases of gastric carcinoma, one case of colon cancer, and one case of adenocarcinoma of unknown primary in which the patient developed a syndrome analogous to thrombotic thrombocytopenic purpura, consisting of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure without definite evidence of disseminated intravascular coagulation. In contrast to previous reports, postmortem examination in three of the cases revealed no recurrence or only microscopic foci of residual tumor. In the remaining three, there was clinical and pathologic evidence of grossly disseminated carcinoma. Also in contrast to previous cases, all patients evidenced azotemia and proteinuria at the onset of the syndrome and ultimately uremia was a contributing cause of death. Coagulation profiles showed prolonged thrombin times and elevated fibrin degradation products in four instances and did not distinguish the patients with grossly metastatic disease from those with no tumor or only microscopic residua. Circulating immune complexes containing carcinoembryonic antigen were found in the patient with metastatic colon carcinoma. The syndrome was clinically identical whether or not grossly metastatic tumor was present, and it should not be attributed to advanced disease without definite clinical or pathologic evidence of a recurrence.

Prolonged Thrombin Time Probably No Contraindication to Kidney Biopsy

Prolonged Thrombin Time Probably No Contraindication to Kidney Biopsy

Acquired dysfibrinogenemia in a hemophiliac with hepatoma: Resolution of fibrinogen dysfunction following chemotherapy

A 17-year-old male with previously undiagnosed congenital Factor IX deficiency (13%) presented with gastrointestinal bleeding and a hepatic mass. Prolonged thrombin and Reptilase times, which partially corrected with CaCl2 and a discrepancy between thrombin-clottable and immunoreactive plasma fibrinogen, suggested a dysfibrinogenemia. Laparotomy disclosed metastatic hepatoma. Adequate hemostasis was obtained with clotting factor replacement, but wound healing was delayed. Patient fibrinogen purified with 2.1 M glycine migrated normally on immunoelectrophoresis and 7.5% polyacrylamide-SDS gel electrophoresis. However, fibrin monomers prepared from purified patient fibrinogen displayed impaired aggregation at high and low ionic strengths when compared with fibrin monomers from normal and control Factor IX deficient subjects. Aggregation of normal monomers was delayed when mixed 1:1 with patient monomers. Fibrinopeptide release was normal, and total sialic acid content was similar to that of normal and control fibrinogens. Chemotherapy, consisting of 5-FU given via intra-arterial hepatic infusion, was accompanied by significant transient clinical improvement which coincided with correction of thrombin clotting times and fibrin monomer aggregation. Reappearance of fibrinogen dysfunction occurred with clinical deterioration prior to death from metastatic hepatoma and sepsis. This case is the first to corroborate the postulated tumor marker role of dysfibrinogenemia in a patient with hepatoma by documenting a direct relationship with response to chemotherapy.

Disseminated intravascular coagulation: a clinical/laboratory study of 48 patients.

In summary, this series of 48 patients with acute and chronic DIC demonstrates the reliability of laboratory tests in both aiding a diagnosis of DIC and in offering reasonable predictability of efficacy of therapy, as noted by the correction of abnormalities after delivery of antiprocoagulant therapy for this syndrome. It appears that the diagnostic tests most likely to aid in diagnosis and to reliably inform the clinician when the intravascular clotting process has been stopped are those that determine the antithrombin-III level, the presence of soluble fibrin monomer, and the finding of elevated fibrin(ogen) degradation products, thrombocytopenia and a prolonged thrombin time in the face of the appropriate type of bleeding in the appropriate clinical setting. In addition, it would appear that mini-dose heparin therapy is highly effective in controlling the intravascular clotting process in acute DIC, whereas antiplatelet therapy utilizing two agents is effective in chronic DIC. In addition, in this population, patients with acute disease demonstrated a 74 percent survival rate and those with chronic disease had a 100 percent survival rate from the disseminated intravascular clotting process.