Differential effect of soluble fibrinogen as a neutrophil activator

A fundamental paradigm involved in acute inflammatory responses to invading pathogens and tissue damage is the migration of specific leukocyte populations to the affected tissues to mount an initial innate response to the aggression. The recruitment of polymorphonuclear neutrophils (PMNs) from the blood is a central event in this respect. The aim of this study was to understand whether fibrinogen is able to modulate the pattern of neutrophil activation and thus contribute to neutrophil recruitment. We demonstrated that fibrinogen induces free radical production by neutrophils without modifying the activation status of Mac-1 (αMβ2, CD11b/CD18), the previously identified neutrophil receptor for fibrinogen. This data indicates that fibrinogen must have an additional different binding site in the neutrophil membrane. Importantly, we propose that as Mac-1 activation was not affected by the binding of fibrinogen, activated neutrophils can further maintain their ability to marginate, roll and adhere to the endothelial walls.     

The intrinsic pathway of coagulation is essential for thrombus stability in mice

Blood coagulation is a highly regulated process involving interactions between platelets, plasma coagulation factors, and the vessel wall. During coagulation in vivo, fibrin formation is thought to be initiated when plasma factor VIIa forms a complex with the membrane protein tissue factor. Coagulation factor XII (FXII, Hageman factor) is required for some in vitro coagulation systems; however, FXII deficiency is not associated with hemorrhage, leading to the conclusion that it is not necessary for hemostasis. We generated FXII-deficient mice to study the contributions of FXII to thrombosis and hemostasis in arterial injury models and in models of acute arterial occlusion. FXII-deficient mice do not experience excessive injury-related bleeding; however, intravital fluorescence microscopy and blood flow measurements in three separate arterial beds revealed a severe defect in formation and stabilization of platelet-rich occlusive thrombi induced by different methods of injuries. Similar findings were observed for mice deficient in factor XI, a substrate of activated FXII. Infusion of human FXII into FXII null mice restored thrombus formation. These findings demonstrate that FXII-mediated fibrin formation is crucial for pathological arterial thrombosis but not for hemostasis and suggest that FXII could be an ideal target for safe anticoagulation.     

In vitro evaluation of clot quality and stability in a model of severe thrombocytopenia: effect of fibrinogen,factor XIII and thrombin-activatable fibrinolysis inhibitor

Background

The treatment options in severe thrombocytopenia (platelet count ≤20×10⁹/L) are limited. The aim of this study was to investigate ways of improving blood clotting and stability in reconstituted thrombocytopenia.

Materials and methods

Thrombocytopenia (platelets [16±4]×10⁹/L) was created by differential centrifugation of normal blood followed by reconstitution of whole blood which was subjected to clotting in a rotation thromboelastometer by CaCl₂ and tissue factor, and to fibrinolysis by tissue plasminogen activator (tPA). In separate experiments, blood was diluted by 40% with TRIS/saline solution. Blood was treated with fibrinogen (fib), factor XIII (FXIII), and thrombin-activatable fibrinolysis inhibitor (TAFI).

Results

The maximum clot firmness of thrombocytopenic blood was approximately 2-fold less than that of intact blood. Supplementation of blood with fib and FXIII improved clot formation. In the presence of tPA, among fib, FXIII and TAFI, only fib stimulated clot propagation whereas each of these agents increased clot strength. There was a synergistic effect when fib was added together with FXIII or TAFI. Fibrinolysis was inhibited by TAFI and to a greater extent by TAFI + FXIII. Fourty percent dilution of blood reduced clot strength and increased susceptibility to tPA. Clot strength was increased by the treatments in the following order: fib/FXIII/TAFI > fib/TAFI > fib > TAFI > FXIII. In the presence of tPA, TAFI and FXIII lysed the clots significantly more slowly. This effect was stronger when blood was treated with the combination of fib/FXIII/TAFI. Doubling the fib concentration, alone or together with other agents, did not improve clot strength or stability.

Discussion

Augmentation of clot formation and anti-fibrinolysis by combining fib, FXIII and TAFI may be beneficial for the treatment of patients with severe thrombocytopenia especially when complicated by haemodilution following introduction of fluids to compensate for massive blood loss.     

Formation of left ventricular thrombus in acute myocardial infarction: significance of the determination of fibrinogen, of products of fibrinogen degradation, and of plasminogen]

OBJECTIVE: To evaluate the significance of the fibrinogen, the plasminogen and the fibrinogen degradation products levels as marks of left intraventricular thrombosis (LIVT) in acute myocardial infarction (AMI). METHODS: 219 consecutive patients of AMI admitted in a Coronary Care Unit of an University Hospital, were prospectively studied. All protocols included a clinical evaluation, an M-mode and 2D echocardiographic study and blood samples, at day 1, 3, 7 and at hospital discharge. In the intraventricular thrombus evaluation just the 4 Asinger grade was considered. In the laboratory evaluation we used: the Clauss chronometric method for the fibrinogen, the colorimetric method for the plasminogen and the agglutination in plaque for the FDP. The patients with ECO in the 2 or 3 Asinger grades and those in which ECO and laboratory study were not performed in the same day, were excluded. 101 patients remained on the study, and they were divided in two groups: 53 patients with LIVT and 48 patients without it. RESULTS: In both groups the fibrinogen raised along the first six days of the AMI, however in the group with LIVT this level didn't raise as high as in the group without LIVT (p < 0.001). In the FDP evaluation two peaks were found, one at 48 hours and another on the 6 th day, but there were no differences between the two groups. The plasminogen values raised along the first week of AMI, in a similar way in both groups. CONCLUSIONS: a) Fibrinogen levels raises in AMI, but this elevation is significantly smaller in the group with LIVT, which suggests fibrinogen consume in fibrin formation of the thrombus. b) FDP and plasminogen levels raise along the first week of AMI, but in a similar way in the two groups. c) None of these parameters permitted to individualize patients with thrombus formation.     

Antibody blockade or mutation of the fibrinogen gamma-chain C-terminus is more effective in inhibiting murine arterial thrombus formation than complete absence of fibrinogen

An elevated plasma fibrinogen level is a risk factor for thrombotic cardiovascular disease, but which of fibrinogen's functions is responsible for the increased risk is unknown. To define better the contribution of fibrinogen to large vessel thrombus formation, we studied carotid artery thrombosis in wild-type mice, mice lacking fibrinogen (fbg^–/–), mice treated with 7E9 (a blocking antibody to the fibrinogen -chain C-terminus), and mice expressing a mutant fibrinogen (5) that lacks the -chain platelet-binding motif QADGV. In control mice, thrombus formation resulted in occlusion in 8 ± 2 minutes (mean ± SD). In fbg^–/– mice, thrombi grew to large sizes, but then they abruptly embolized, confirming previous observations by others in an arteriolar thrombus model. In contrast, mice treated with 7E9 and 5 mice developed only small, nonoclusive mural thrombi and embolization was limited. These findings reveal that a fibrinogen antibody, 7E9, or a fibrinogen mutant retaining clotting function, can limit thrombus formation more effectively than the complete absence of fibrinogen. We hypothesize that the smaller thrombi in these animals result from the ability of fibrin to bind and sequester thrombin and/or the ability of the altered fibrinogen molecules, which cannot recruit platelets, to bind to and passivate the surface.      

Laser-induced thrombus formation in mouse brain microvasculature: effect of clopidogrel

Antiplatelet drugs have been evaluated by measuring platelet aggregation ex vivo, but in vivo studies were scanty. The purpose of this study was to observe the effects of an antiplatelet agent (clopidogrel) on the process of laser-induced thrombus formation in mice using intravital fluorescence microscopy. C57 BL/6J mice (n = 19) were anesthetized using chloral hydrate. The head of each mouse was fixed with a head holder, and a cranial window was made in the parietal region. Platelets were labeled in vivo by intravenous administration of carboxyfluorescein diacetate succinimidyl ester. Clopidogrel (1 mg/kg, n = 6; 10 mg/kg, n = 6) was administered orally for 2 days before the experiment. Another seven mice were used as controls. Laser irradiation (1,000 mA, 9.8 mW, diode-pumped solid-state (DPSS) laser 532 nm) was directed for 4 s at pial arteries to induce thrombus formation. Labeled platelets and thrombus were observed continuously under fluorescence microscopy. We recorded the area of thrombus after 30 min and determined the complete occlusion rate. After laser irradiation to the pial artery, complete occlusion rate was significantly lower in the clopidogrel (10 mg/kg) group (16%, 4/25 vessels) than in the control group (60%, 12/20 vessels) or clopidogrel (1 mg/kg) group (55%, 11/20 vessels). Area of platelet thrombus at 30 min after laser irradiation was significantly smaller in the clopidogrel (10 mg/kg) group (209 ± 128 μm(2)) than in the control group (358 ± 256 μm(2)) or clopidogrel (1 mg/kg) group (355 ± 57 μm(2)). The apparatus which we developed is convenient for inducing thrombus formation by causing endothelial cell damage to the brain surface vasculature in small animals without damage of extravascular tissue. Clopidogrel significantly inhibited laser-induced thrombus formation in pial arteries of mice in a dose-dependent manner.     

The hemostatic effect of bovine peptide-B from fibrinogen

Bovine peptide-B from fibrinogen was active in the hemostasis of rat tail arterioles. The time of bleeding exhibits an inverse log proportionality to the concentration of bovine peptide-B. The peptide produced a 10-fold decrease in the time of bleeding at the highest concentration tested. Prolonged incubation of the peptide with the system was unnecessary and it appeared to participate immediately as a hemostatic agent. These findings suggest that hemostasis was due to vasoconstriction since coagulation time remained constant as the bleeding time decreased with increasing concentration of peptide-B. Bovine peptide-B is interpreted as a physiological substance which probably acts on some smooth muscle receptor.     

Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect

Highly purified pro-urokinase (pro-UK) or single-chain urokinase-type plasminogen activator (scu-PA) was treated with diisopropylfluorophosphate (1 mmol/L) to eliminate traces of two-chain UK activity. This preparation was found to retain a low activity against a chromogenic substrate (S2444), equivalent to 0.1% to 0.5% of the activity of its plasmin-activated derivative. Evidence is presented that the intrinsic activity of pro-UK (scu-PA) was sufficient to activate plasminogen on a fibrin plate or in buffer and was far more reactive against Lys-plasminogen than against Glu-plasminogen. The relative resistance of Glu-plasminogen to activation was overcome by the addition of lysine (25 mmol/L) to the reaction mixture. By contrast, in plasma, pro-UK (scu-PA) was stable and nonreactive for greater than 72 hours when incubated (37 degrees C). Pro-UK (scu-PA) did not form sodium dodecyl sulfate-stable inhibitor complexes, whereas complexation occurred rapidly with UK. Only at high concentrations of pro-UK (scu-PA) (greater than or equal to 250 IU/mL) did plasminogen activation in plasma occur. The relative inertness of pro-UK (scu-PA) in plasma, in contrast to its low-grade enzymatic activity in buffer, was attributed to the effect of inhibitors. The addition of EDTA or the removal of divalent cations by dialysis was associated with a lower threshold for nonspecific plasminogen activation by pro-UK (scu-PA) in plasma. Replacement of Ca++ but not other cations restored baseline conditions. In the presence of a clot, fibrin-selective plasminogen activation and clot lysis were triggered. Lysis was accompanied by less than 10% conversion of pro-UK (scu-PA) to two-chain UK, suggesting that the intrinsic activity of pro-UK (scu-PA) itself may have been responsible for fibrinolysis, although a contribution by the small amount of UK generated could not be excluded. Similarly, pro-UK (scu- PA) supported clot lysis for several days in the same plasma before the effect dissipated as a result of degradation to UK. When Glu- plasminogen in plasma was replaced by Lys-plasminogen, or when lysine was added to normal plasma, nonselective plasminogen activation and fibrinogenolysis occurred. It was concluded that under the experimental conditions, the fibrin specificity of pro-UK (scu-PA) can be explained by its selective activation of fibrin-bound plasminogen and is due to the latter's Lys-plasminogen-like conformation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Urokinase therapy for a central venous catheter thrombus

A 670 g premature infant is described in whom an intracardiac thrombus was documented. This thrombus formation probably resulted as a complication of an indwelling right atrial catheter. Thrombolytic therapy with urokinase was instituted, resulting in total and rapid dissolution. No hemorrhagic complications resulted. We believe that this particular thrombolytic therapy is safe and effective and should be considered when facing this particular complication.     

Evolutionary stability of fibrinogen epitopes implicated in fibrin polymerization and in fibrinolysis

In this work, fibrinogen evolution was analysed by testing the reactivity of fibrinogen from different species with monoclonal antibodies against human fibrinogen fragment D. One epitope concerning the fibrin polymerization site 'a' and two epitopes responsible for tPA binding to fibrin were conserved in all mammalian fibrogens tested but not in crab coagulogen or pleurodella fibrinogen. In these two species, some epitopes which are not implicated in fibrinogen function were conserved. Therefore, we can conclude that polymerizing site 'a' and tPA binding sites have not been modified for at least 80 million years.