Functional Fibrinogen Levels Research Articles

Electrospray ionization mass spectrometry identification of fibrinogen Banks Peninsula (gamma280Tyr-->Cys): a new variant with defective polymerization.

Fibrinogen Banks Peninsula was identified in the mother of a patient referred for investigation following recurrent epistaxis. Coagulation tests revealed prolonged thrombin and reptilase times and a decreased functional fibrinogen level. Thrombin-catalysed release of fibrinopeptides A and B was normal, and no abnormalities were detected by DNA sequencing of the regions encoding the thrombin cleavage sites in the Aalpha and Bbeta genes. Reducing SDS-PAGE and reverse-phase HPLC analysis of purified fibrinogen chains were normal, as was electrospray ionization mass spectrometry (ESI-MS) analysis of isolated Aalpha and Bbeta chains. However ESI-MS revealed a mass of 48345 D for the isolated gamma chains, 31 D less than the measured mass of control chains (48376 D). Since normal and abnormal gamma chains were not resolved, this implies a 60-62 D mass decrease in 50% of the molecules. A 60 D decrease was confirmed when DNA sequencing indicated heterozygosity for a mutation of Tyr-->Cys at codon 280 of the gamma chain gene. Fibrin monomer polymerization revealed a delayed lag phase and reduced final turbidity and although factor XIIIa crosslinking of fibrinogen was normal, it is likely that this delay is due to impaired D:D self association. Recent crystallographic studies show residues gamma280 and gamma275 make contact across the D:D interface, suggesting a similar mechanism for the polymerization defects in fibrinogens Banks Peninsula and Tokyo II (gamma275Arg-->Cys).

Elevated plasma fibrinogen: cause or consequence of cardiovascular disease?

An association between increased plasma fibrinogen and an increased risk for myocardial infarction (MI) is well established, but the nature of this association is subject to debate. Our aim was to shed light on the potentially causal nature of this association. We examined whether increased plasma fibrinogen, due to a condition that is independent of cardiovascular events, also increases the risk for MI. A case-control study was performed in 139 subjects with a history of MI and 287 control subjects selected from the Rotterdam Study, a population-based cohort of 7983 subjects aged 55 years and older. The genotype of the -455G/A polymorphism in the fibrinogen beta-gene was determined by polymerase chain reaction. Functional plasma fibrinogen levels were determined according to von Clauss. The plasma level of fibrinogen was significantly higher in subjects with one or two A alleles compared with subjects with the GG genotype: 3.8 (95% confidence interval [CI], 3.6 to 3.9) g/L and 3.6 (3.5 to 3.7) g/L, respectively. With increasing plasma fibrinogen level, the risk for MI increased gradually; a rise in fibrinogen of 1 g/L was associated with a 45% increased risk (odds ratio adjusted for age, sex, and smoking, 1.45; 95% CI, 1.12 to 1.88). There was no association between the genotype of the -455G/A polymorphism and the risk for MI. The -455G/A polymorphism is therefore associated with increased plasma fibrinogen levels but not with an increased risk for MI. These findings indicate that an increased plasma fibrinogen level due to this genetic factor does not increase the risk for MI.

Effects of Gemfibrozil and Ciprofibrate on Plasma Levels of Tissue-type Plasminogen Activator, Plasminogen Activator Inhibitor-1 and Fibrinogen in Hyperlipidaemic Patients

Evaluation of fibrate treatment in humans has focused primarily on its anti-lipidaemic effects. A potentially favourable haemostasis-modulating activity of fibrates has also been recognized but the data are not consistent. We sought to learn more about this variability by examining the effects of gemfibrozil and ciprofibrate on plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and fibrinogen in primary hyperlipidaemic patients after six and twelve weeks of treatment using different assay systems for PAI-1 and fibrinogen. Although both fibrates effectively lowered triglyceride and cholesterol levels, no effect on the elevated baseline antigen levels of t-PA and PAI-1 was observed after fibrate treatment. However, both fibrates influenced plasma fibrinogen levels, albeit in a different way. Fibrinogen antigen levels were elevated by 17.6% (p <0.05) and 24.3% (p <0.001) with gemfibrozil after six and twelve weeks, respectively, whereas with ciprofibrate there was no effect. Using a Clauss functional assay with either a mechanical end point or a turbidity-based end point, no significant change in fibrinogen levels was seen after six weeks of gemfibrozil treatment. However, after twelve weeks, gemfibrozil enhanced functional fibrinogen levels by 7.2% (p <0.05) as assessed by the Clauss mechanical assay, but decreased functional fibrinogen levels by 12.5% (p <0.0001) when a Clauss assay based on turbidity was used. After six or twelve weeks of ciprofibrate treatment, functional fibrinogen levels were decreased by 10.1% (p <0.001) and 10.5% (p <0.0001), respectively on the basis of Clauss mechanical and by 14.2% (p <0.001) and 28.2% (p <0.0001), respectively with the Clauss turbidimetric assay. A remarkable and consistent finding with both fibrates was the decrease in functionality of fibrinogen as assessed by the ratio of functional fibrinogen (determined by either of the two Clauss assays) to fibrinogen antigen. Taken together, our results indicate that at least part of the variability in the effects of fibrates on haemostatic parameters can be explained by intrinsic differences between various fibrates, by differences in treatment period and/or by the different outcomes of various assay systems. Interestingly, the two fibrates tested both reduced the functionality of fibrinogen.

7. Modulation of plasma fibrinogen levels by medication

Elevated plasma fibrinogen levels represent an increased risk for cardiac events. This has enhanced the interest in identifying agents that can normalize elevated plasma fibrinogen levels. Agents that have this capacity are the lipid lowering fibric acid derivatives (e.g. ciprofibrate) and the platelet aggregation inhibitor ticlopidine. Fibrinogen is a very heterogenous molecule that has a number of different functions. Fibrinogen is an important protein in the coagulation process. Besides that, the concentration of fibrinogen is the main determinant of the plasma viscosity. The functionality of fibrinogen is partly determined by the molecular weight (MW) form of fibrinogen: the high MW (HMW) form clots faster than the low and low' (LMW and LMW) forms. The mechanism of the fibrinogen decrease by fibrates and ticlopidine has not yet been elucidated and, therefore, we studied the effects of treatment with these drugs on functional fibrinogen, fibrinogen antigen and (HMW+LMW) levels. The effect of ticlopidine was studied in 26 healthy volunteers and in 26 patients with stable angina pectoris. The effect of ciprofibrate was studied in 51 hypercholesterolemic patients. In the healthy volunteers, the mean (SD) baseline level of functional fibrinogen was 2.35 g/L (SD 0.35), for fibrinogen antigen this was 2.49 g/L (SD 0.63), and for (HMW+LMW) fibrinogen 1.97 g/L (SD 0.53). After 4 weeks of ticlopidine administration, the functional fibrinogen level had decreased with 0.20 g/L (9%, p=0.005 using the paired Student t-tesl), whereas the fibrinogen antigen levels had not significantly changed. In the stable angina patients, the mean (SD) baseline levels of functional plasma fibrinogen was 3.44 g/L (SD 0.61), fibrinogen antigen 2.60 g/L (SD 0.49) and for (HMW+LMW) fibrinogen 2.70 g/L (SD 0.60). These baseline fibrinogen levels were significantly higher than in the volunteer group. After four weeks ticlopidine administration the functional fibrinogen levels had decreased with 0.39 g/L (11%, p<0.005), whereas the fibrinogen antigen and (HMW+LMW) fibrinogen levels had not significantly changed. In the hypercholesterolemic group, the mean (SD) baseline level of functional fibrinogen was 3.38 g/L (SD0.61), for (HMW+LMW) fibrinogen this was 3.04 (SD 0.80). After 12 weeks of treatment with ciprofibrate, the functional fibrinogen levels were significantly decreased to 2.42 g/L (SD 0.42) (p<0.0005), whereas the (HMW+LMW) fibrinogen levels had not significantly changed. Therefore, the fibrinogen lowering effect of ticlopidine and ciprofibrate are likely to be a modulation of the functionality of the molecule. © Pearson Professional Ltd 1996.

40. Modulation of plasma fibrinogen levels by ticlopidine in healthy volunteers and patients with stable angina pectoris

Elevated plasma fibrinogen levels represent an increased risk for cardiac events. Ticlopidine is a drug that inhibits the ADP-induced aggregation of blood platelets and it also has been described that ticlopidine can decrease the plasma fibrinogen level in patients with vascular diseases. The mechanism of this decrease has not yet been elucidated and therefore mechanisms that are known to affect fibrinogen levels were studied, viz. the acute phase reaction, total fibrin and fibrinogen degradation (TDP) levels and the fibrinogen G/A4 -gene polymorphism. The fibrinogen lowering effect of ticlopidine was studied in 26 healthy volunteers and in 26 patients with stable angina pectoris in a double blind, randomized cross-over study versus placebo. Functional plasma fibrinogen levels were measured with the Clauss assay and antigen levels with an enzyme immunoassay. C-reactive protein (CRP) and TOP levels were measured with an enzyme immuno assay. In the healthy volunteers the mean (SD) baseline level of functional plasma fibrinogen was 2.35 g/L (SD 0.35) and for fibrinogen antigen this was 2.49 g/L (SD 0.63). The geometrical mean (central 95% range) of CRP as 0.21 mg/L (0.02-2.36) and for TOP this was 0.18 ng/mL (0.03-1.00). After 4 weeks of ticlopidine administration, the functional fibrinogen levels had decreased with 0.20 g/L (9%, p=0.005 using the paired Student t-test) whereas the fibrinogen antigen levels, the CRP and the TOP levels had not significantly changed. In the stable angina pectoris patient the mean (SD) baseline levels of functional plasma fibrinogen were 3.44 g/L (SD 0.61) and of fibrinogen antigen they were 2.60 g/L (SD 0.49). The geometrical mean (central 95% range) of CRP was 1.45 mg/L (0.15-14.44) and for TOP this was 0.28 ng/mL (0.05-1.62). These baseline fibrinogen, CRP and TOP levels were significantly higher than in the volunteer group. After four weeks ticlopidine administration the functional fibrinogen levels had decreased with 0.39 g/L (11%, p<0.005), whereas the fibrinogen antigen, the CRP and the TDP levels had not significantly changed. The functional and antigen levels of fibrinogen, CRP and TDP did not change significantly during the placebo period in the volunteers or the patients. Neither in the volunteers nor in the patients was the effect of ticlopidine on the fibrinogen levels associated with the fibrinogen G/A~455 genotype. Therefore, the fibrinogen lowering effect of ticlopidine is likely to be a modulation of the functionality of the molecule and unlikely to be modulated by the acute phase reaction, TDP-levels or the fibrinogen β-gene polymorphisms. © Pearson Professional Ltd 1996.

24. Quantitative and functional fibrinogen levels in cardiovascular disease in relation to the genetical status

24. Quantitative and functional fibrinogen levels in cardiovascular disease in relation to the genetical status

127. Levels of functional versus intact fibrinogen in 140 patients with acute myocardial infarction treated with Reteplase

Reteplase, the novel K2P-deletion mutant of t-PA, was administered in a dose-finding multicenter study (GRECO) to 140 patients (pts) with acute myocardial infarction (AMI). 40 pts (group A) received an 18 mg (10 Megaunits (MU)) i.V. single bolus of Reteplase and 100 subsequent pts (Group B) were treated with a 27 mg (15 MU) i.v. single bolus. Fibrinogen (fbg) was measured using two different methods before (pre) and 2, 4, 8 and 24 h after Reteplase: the first fbg method was a clotting rate assay according to the Clauss method (functional fbg), the second an enzyme immunoassay (EAI) for immunologically intact fbg. In 10 out of the group B pts (group C; 27 mg) changes of fbg levels were measured in very close intervals until 18 days (dl8) after thrombolysis. Results: The levels of functional (Clauss) and intact (EIA) fbg differed significantly at each time point: A (Clauss/EIA; median values; [g/1]): Pre: 2.8/3.5; 2 h: 1.7/2.7 (60%/77% of the pre-bolus value of each method); 4 h: 1.7/2.7 (62%/76%); 8 h: 1.5/2.6 (54%/74%); 24 h: 2.2/3.0 (79%/86%) (p<0.001 between Clauss and EIA at any time of sampling). B: Pre: 2.6/3.2; 2 h: 1.0/2.1 (37%/66%); 4 h: 0.9/2.2 (36%/69%); 8 h: 1.0/2.2 (37%/69%); 24 h: 1.6/2.4 (63%/75%) (p<0.0005). C: Pre: 2.7/3.1; 30 min.: 1.6/2.5; 60 min.: 1.3/2.4; 2 h: 1.2/2.3; 4 h: 1.2/2.2 (44%/69%); 8 h: 1.3/2.2; 24 h: 1.8/2.5; 48 h: 2.8/2.7; 72 h: 4.1/3.6; d6: 4.7/3.8 (173%/123%); d9: 4.5/3.9 (167%/126%); d!2: 4.1/3.5; d!5: 3.9/3.2; d!8: 4.0/3.4 (p<0.05; exception: 48 h: not significant). Conclusions: The levels of functional (Clauss) fbg showed lower nadirs and higher rebound levels (C) compared to the intact (EAI) fbg levels. Lower nadirs measured by the Clauss method might reflect the disturbance of this method by fibrin(ogen) degradation products. Higher rebound levels of functional fbg (Clauss) might indicate newly synthesized fast clotting HMW (high molecular weight) fbg and therefore a risk factor for reocclusions in AMI pts treated with thrombolytic agents. In contrast to our previous studies in rt-PAtreated AMI pts, the pretreatment ratio of functional (Clauss) to intact (EAI) fbg here was <0.9 in groups A, B and C compared to 1.6 in the rt-PA study. © Pearson Professional Ltd 1996.

Modulation of Plasma Fibrinogen Levels by Ticlopidine in Healthy Volunteers and Patients with Stable Angina Pectoris

Elevated plasma fibrinogen levels are associated with an increased risk for cardiac events. Ticlopidine is a drug that inhibits the ADP-induced aggregation of blood platelets and it also has been described that ticlopidine can decrease the plasma fibrinogen level in patients with vascular diseases. The mechanism of this decrease has not yet been elucidated and therefore mechanisms that are known to affect fibrinogen levels were studied, viz, the acute phase reaction, total fibrin plus fibrinogen degradation (TDP) levels and the polymorphisms of the fibrinogen beta-gene. The fibrinogen lowering effect of ticlopidine was studied in 26 healthy volunteers, selected on genotype of the Bcl] polymorphism of the fibrinogen beta-gene, and in 26 patients with stable angina pectoris in a double blind, randomized cross-over study. Functional plasma fibrinogen levels were measured with the Clauss assay. Fibrinogen antigen, C-reactive protein (CRP) and TDP levels were measured using an enzyme immuno assay (EIA). In the healthy volunteers the functional fibrinogen levels had decreased by 0.20 g/l (9%, p = 0.005 using the paired Student l-test) after 4 weeks of 250 mg bid ticlopidine administration, whereas fibrinogen antigen, CRP and TDP levels were not significantly changed. In the stable angina pectoris patients the pre-treatment fibrinogen, CRP and TDP levels were significantly higher than in the volunteer group. After four weeks 250 mg bid ticlopidine administration the functional fibrinogen levels had decreased by 0.38 g/l (11%, p < 0.005), whereas the fibrinogen antigen, CRP and TDP levels were not significantly changed. The levels of functional and antigen fibrinogen, CRP and TDP did not change significantly during the placebo period in the volunteers or the patients. Neither in the volunteers nor in the patients was the effect of ticlopidine on the fibrinogen levels associated with the fibrinogen beta-gene polymorphisms. Therefore, the fibrinogen lowering effect of ticlopidine is likely to be a modulation of the functionality of the molecule and unlikely to be modulated by the acute phase reaction, TDP-levels or the fibrinogen beta-gene polymorphisms.

Sensitivity of experimental venous and arterial thrombosis and bleeding to ancrod-induced defibrinogenation

The effect of ancrod-induced defibrinogenation on thrombosis and bleeding time was determined in anesthetized rats. Functional plasma fibrinogen levels were reduced 42, 71, 94 and 93% by ancrod doses of 5, 10, 20 and 30 U/kg, respectively, while a 2.5 U/kg dose was without significant effect. Ancrod inhibited vena cava thrombosis induced by partial stasis of blood flow combined with mild vascular injury. Thrombus weight was decreased 85 and 93% by the 10 and 20 U/kg doses, but was unaffected at lower doses. In contrast, ancrod doses of up to 30 U/kg did not significantly decrease carotid artery thrombi formed in response to oxidative transmural vessel injury. Ancrod caused a dose-dependent increase in bleeding time measured by puncturing small mesenteric arteries with a hypodermic needle. The bleeding time increase was ∼38% in response to the 2.5 and 5 U/kg doses, and 182% in response to the 10 U/kg dose. These studies demonstrate that ancrod-induced reductions in plasma fibrinogen more effectively inhibit venous compared to arterial thrombosis, although these activities require doses that also increase bleeding time in small arteries.

Vampire Bat Salivary Plasminogen Activator Promotes Robust Lysis of Plasma Clots in a Plasma Milieu Without Causing Fluid Phase Plasminogen Activation

Vampire bat salivary plasminogen activator (BatPA), human tissue-type plasminogen activator (tPA) or streptokinase (SK) were incubated in human citrated plasma containing a plasma clot that was radiolabelled with iodine-125 fibrin(ogen). Complete clot dissolution by BatPA (30 nM) was associated with slight activation of "fluid phase" plasminogen; the plasma levels of functional fibrinogen and alpha 2-antiplasmin decreased by only 8 and 19%, respectively. Addition of SK (3,600 IU/ml) to the clot-containing plasma caused complete clot lysis and massive activation of the "fluid phase" plasminogen, leading to greater than 60 and 96% decreases of the functional levels of fibrinogen and alpha 2-antiplasmin, respectively. Incubation of tPA (30 nM) in clot-containing plasma caused complete clot lysis as well as substantial activation of "fluid phase" plasminogen; the plasma levels of functional fibrinogen and alpha 2-antiplasmin decreased by 45 and 79%, respectively. The profound degradation of fibrinogen in the SK and tPA but not BatPA-containing samples was confirmed by immunoblot analysis. Additional experiments showed that the presence of soluble clot lysate in plasma containing tPA enhanced the extent of fibrinogen degradation from 25% to greater than 60%; the addition of soluble clot lysate to the plasma containing BatPA did not prompt further fibrinogen degradation. Finally, studies using exogenous alpha 2-antiplasmin suggested that plasmin generated via tPA-mediated activation of "fluid phase" plasminogen does not play an important role in clot dissolution.

Pharmacodynamics of thrombolysis with recombinant tissue-type plasminogen activator. Correlation with characteristics of and clinical outcomes in patients with acute myocardial infarction. The TAMI Study Group.

Coagulation analysis was performed on blood samples from 386 patients with acute myocardial infarction drawn before, during, and after a continuous intravenous infusion of 150 mg recombinant tissue-type plasminogen activator (rt-PA) (Activase). Plasma rt-PA rose to peak levels of 2.1 +/- 3.1 micrograms/ml (mean +/- SD). Fibrinogen levels measured by coagulation rate and by sulfite precipitation decreased from baseline levels of 3.0 +/- 0.9 and 3.2 +/- 1.0 g/l, respectively, to nadir levels of 1.4 +/- 0.75 and 1.8 +/- 0.92 g/l, respectively, and were associated with peak levels in serum of fibrinogen-degradation products (FDP) of 230 +/- 470 micrograms/ml. Forty percent of patients experienced a nadir functional-fibrinogen level of less than 1.0 g/l, whereas 20% fell below 0.5 g/l. Nadir fibrinogen levels did not correlate with patency of the infarct-related coronary artery at 90 minutes or with risk of coronary vessel reocclusion within 7-10 days. However, the risk of coronary artery reocclusion was inversely related to the baseline functional fibrinogen level (p = 0.0008), with the magnitude of its drop to nadir level (p = 0.0003) as well as to peak levels of FDP (p = 0.038). Quantitative blood loss correlated with all markers for systemic fibrinogenolysis including nadir fibrinogen level (r = -0.20, p = 0.0011), percent decrease of fibrinogen (r = 0.22, p = 0.001), and peak FDP levels (r = 0.14, p = 0.020). Both patients who experienced intracranial hemorrhage presented with high baseline fibrinogen levels and experienced extensive degradation of coagulable fibrinogen. Overall, patients at greatest risk of systemic fibrinogenolysis tended to be relatively older women with lower body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring of Hemostasis Parameters During Coronary Thrombolysis with Recombinant Tissue-Type Plasminogen Activator

The monitoring of changes in the blood coagulation and fibrinolytic systems during thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) may be complicated by artifacts due to in vitro activation after blood collection and to interference of other agents (e.g., heparin) in the assays. In 106 patients with early acute myocardial infarction, infused with 150 mg of rt-PA (G11044) intravenously over 5 to 8 hours, blood samples were collected into liquid citrate supplemented with the plasmin inhibitor aprotinin (200 KIU/ml plasma) or on a lyophilized mixture of acidified citrate and the synthetic t-PA inhibitor D-Phe-Pro-Arg-CH2Cl (PPACK). A good correlation between precipitable (sulphite) and functional (clotting rate) fibrinogen levels was observed in plasma collected on citrate before therapy (r = 0.76) and in samples collected after 3 hours on either aprotinin (r = 0.87) or PPACK (r = 0.82). Precipitable fibrinogen levels were approximately 10% higher than functional level, in baseline samples collected on citrate alone and approximately 20% higher in 3 hour samples collected on either PPACK or aprotinin. Fibrinogen levels measured with both assays correlated well, but were somewhat higher in samples collected on PPACK than on aprotinin. rt-PA antigen levels assayed in plasma collected in either inhibitor correlated well (r = 0.90) but were 10-20% higher in PPACK containing samples. Addition of heparin up to 9 units/ml to plasma had no effect on the functional fibrinogen assay.(ABSTRACT TRUNCATED AT 250 WORDS)