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PubMed1979

Detection of fibrin monomer. Comparison of the immune precipitate method with the serial-dilution protamine sulfate test and the ethanol gel test.

In a previous publication, a method for measuring fibrin monomer in plasma with the use of an immune precipitate of fibrinogen was described. The method was found to be more sensitive to unstabilized fibrin monomer than the serial-dilution protamine sulfate test, or the ethanol gel test, and detected fibrin in a mixture of the plasmin digestion products of fibrin. In the present study the sensitivity of the immune precipitate method for detecting specific plasmin digestion products of fibrin X, Y, D, and E, its sensitivity for stabilized fibrin monomer complexes, and its sensitivity for fibrin retaining B-peptide were measured and compared with the corresponding sensitivities of the serial-dilution protamine sulfate test and the ethanol gel test for detecting these same products. The immune precipitate method was found to be highly sensitive to stabilized fibrin monomer complexes and to fibrin retaining B-peptide; to be significantly less sensitive to the plasmin digestion fragments X, Y, and E; and to be insensitive to fragment D. The serial-dilution protamine sulfate test and the ethanol gel test were found to be insensitive to all of the plasmin digestion products of fibrin and less sensitive to the other fibrin complexes.

Batroxobin↗
PubMed2002

Inhibition of fibrin-bound thrombin by a covalent antithrombin-heparin complex.

Numerous studies have shown that fibrin-bound thrombin (IIa) is protected from inhibition by antithrombin (AT) + heparin (H) due to the formation of a ternary fibrin.IIa.H complex. We investigated factors affecting the inhibition of fibrin.IIa by a covalent complex of AT and H (ATH). The rate of IIa reaction with ATH was decreased 2-3-fold by fibrin monomer as compared to 57-fold for AT + heparin with high AT affinity. Furthermore, although the reaction of AT + H with a IIa mutant with decreased H binding (RA-IIa) was inhibited 2-3-fold in the presence of fibrin, reaction rates of ATH + RA-IIa were not reduced by fibrin. The relative difference in the effect of fibrin on the ATH reaction with RA-IIa compared to that for reactions of AT + H with RA-IIa is consistent with the fact that, in the absence of fibrin, the rate of the ATH reaction with RA-IIa relative to IIa was much less reduced (8-fold) compared to the corresponding reactions of AT + H (decreased 306 fold). Similarly, the addition of excess H in the absence of fibrin gave only a small decrease in rate of ATH + IIa reaction. However, in the presence of fibrin, the addition of 40-fold excess H decreased the rate of ATH inhibition of IIa by 1 order of magnitude. Experiments with ATH containing low molecular weight heparin chains with low AT affinity showed that IIa inhibition requires ATH with long chains that activate the AT moiety. Finally, electrophoresis of fibrin +/- ((125)I-)IIa +/- ((125)I-)ATH on native and denaturing gels showed that ATH forms ATH-IIa complexes that remain bound to fibrin through the ATH component. Thus, ATH is a potent inhibitor of fibrin-bound IIa, likely due to the formation of fibrin.ATH-IIa as opposed to fibrin.IIa.H ternary complexes.

Animals↗
PubMed1995

Delivery of demineralized bone powder by fibrin sealant.

The main purpose of this study was to determine whether the use of fibrin sealant as a delivery vehicle for demineralized bone powder would result in bone induction in heterotopic and orthotopic sites. Rat demineralized bone powder alone or in different concentrations of fibrin sealant matrix (4, 8, 15, and 45 mg/ml) was bioassayed for bone induction by implantation in intramuscular sites. Distribution of treatment groups was as follows: demineralized bone powder alone (n = 12), demineralized bone powder plus 4 mg/ml fibrin sealant (n = 11), demineralized bone powder plus 8 mg/ml fibrin sealant (n = 11), demineralized bone powder plus 15 mg/ml fibrin sealant (n = 11), demineralized bone powder plus 45 mg/ml fibrin sealant (n = 10), 4 mg/ml fibrin sealant (n = 13), and 45 mg/ml fibrin sealant (n = 11). In a second group of rats, 8-mm critical-sized calvarial defects were created and treated with demineralized bone powder plus 30 mg/ml fibrin sealant. Intramuscular implants were retrieved after 28 days, while calvarial implants were retrieved at 28 days (n = 8), 3 months (n = 8), or 4 months (n = 5). Implants were then x-rayed and submitted for histology. Results showed bone formation as evidenced by radiopacity and histology. Radiopacity measurements of demineralized bone powder implants alone or in a fibrin sealant matrix were associated with immature woven bone at the implantation site. Fibrin sealant allowed bone formation by demineralized bone powder to occur, improved the handling of demineralized bone powder, and facilitated the shaping of implants.

Animals↗
PubMed1993

Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface.

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (Kd = 3.3 +/- 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 +/- 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 microM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (Kd = 0.66 +/- 0.22 microM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution.

Amino Acid Sequence↗
PubMed2006

Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo.

BACKGROUND: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. OBJECTIVE: Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. RESULTS: The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. CONCLUSIONS: The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing.

Animals↗
PubMed2001

Role of fibrin matrix in angiogenesis.

Angiogenesis, the formation of new blood vessels from existing vessels, plays an important role during development. In the adult, it is limited to the female reproductive system and to tissue repair and pathological conditions. Repair associated angiogenesis is usually accompanied by the presence of inflammatory cells, vascular leakage, and fibrin deposition. The temporary fibrin matrix acts, not only as a sealing matrix, but also as a scaffolding for invading leukocytes and endothelial cells during tissue repair. We have used a three-dimensional fibrin matrix to study the outgrowth of human microvascular endothelial cells in capillary-like tubular structures. This process is induced by the simultaneous addition of an angiogenic growth factor (bFGF or VEGF) and the cytokine TNF alpha, and is enhanced by hypoxia. It involves proteolytic activities, in particular cell bound urokinase/plasmin and matrix metalloproteinase activities. Modulation of the fibrin structure markedly affects the extent and stability of capillary tube formation in vitro. Preparation of fibrin at different pH (7.0-7.8) or crosslinking of the fibrin matrix induces differences in fibrin matrix rigidity and structure. This is accompanied by a change in capillary ingrowth. Heparins, in particular low molecular weight heparins, modulate the fibrin structure and by this action affect angiogenesis in vitro. A mutant fibrinogenNieuwegein, which lacks the terminal part of the A alpha chain of fibrin harboring an RGD sequence and the transglutaminase sequence, provided additional evidence that the structure of fibrin is an important determinant for angiogenesis. These findings may have impact on improving wound healing and on influencing angiogenesis in malignancies with a fibrinous stroma.

Animals↗
PubMedDate not supplied

Unstimulated and thrombin-stimulated platelets binding to immobilized fibrinogen and fibrin on polystyrene supports.

The binding of unstimulated and thrombin-stimulated platelets was studied with immobilized fibrinogen and fibrin on polystyrene. The amount of fibrinogen bound to the polystyrene support was 2 micrograms/tube, which represents 2.35 micrograms/cm2. Immobilized fibrin was obtained by adding thrombin (5 nM) to immobilized fibrinogen. The number of unstimulated 111In-platelets bound to immobilized fibrinogen and fibrin was similar (3.2 +/- 0.3 x 10(6) and 3.1 +/- 0.4 x 10(6) platelets/micrograms fibrin(ogen), respectively). The platelet binding steadily increased. In the first 2 min, the binding rate was 0.23 x 10(6) platelets/micrograms fibrinogen/min. The binding rate then increased rapidly and saturation was reached at 10 min. The extent of the adhesion of resting platelets to immobilized fibrinogen is about one half that of the same platelets stimulated with thrombin. In thrombin-stimulated 111In-platelets, the binding to immobilized fibrinogen and fibrin is time dependent, and saturation is reached at 5 min. The early rate of thrombin-stimulated platelet binding to fibrinogen is about twice that of binding to fibrin (1.25 and 0.74 x 10(6) platelets/micrograms fibrin(ogen)/min, respectively). In saturation conditions, 1 microgram fibrinogen binds 5.7 +/- 0.6 x 10(6) thrombin-stimulated platelets and 1 microgram fibrin binds 4.6 +/- 0.5 x 10(6) thrombin-stimulated platelets. Our results indicate that the rate of platelet aggregation is faster than fibrin formation, and the rate of fibrinogen-platelet binding is faster than that of fibrin-platelet binding. Therefore, after thrombin stimulation, the binding of platelets to fibrin must be secondary to the binding of platelets to fibrinogen.

Blood Platelets↗
PubMed1975

Radioiodinated soluble canine fibrin. Preparation and evaluation as a thrombus localizing agent in the dog.

To develop a thrombus localizing tracer which has characteristics superior to labeled fibrinogen for external detection, we evaluated radioiodinated soluble fibrin. Labeled soluble fibrin was prepared by clotting and dissolving radioiodinated (131I) canine fibrinogen under specified conditions. Biological clearance studies revealed rapid clearance of the labeled soluble fibrin from the blood with a half-life of 5 hours. The accumulation of labeled soluble fibrin and fibrinogen in induced venous thrombi, coronary artery thrombi, and the myocardium was compared in dogs. In venous thrombi, soluble fibrin and fibrinogen exhibited maximum thrombus-blood ratios when they were injected 4 hours after thrombus induction; the thrombus-blood ratio was greater for soluble fibrin than it was for fibrinogen when these agents were injected 4, 8, or 24 hours after thrombosis induction. In induced coronary artery thrombi, soluble fibrin and fibrinogen accumulated to the same extent. Since the blood clearance of soluble fibrin is faster than that of fibrinogen, a higher thrombus-blood ratio was obtained with soluble fibrin in coronary artery thrombi. The thrombus-infarcted myocardium, thrombus-normal myocardium, and infarcted myocardium-normal myocardium ratios obtained with soluble fibrin were slightly higher than those obtained with fibrinogen. Thus, soluble fibrin offers some advantages when it is compared with fibrinogen as a thrombus detecting agent.

Animals↗
PubMed1994

Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion.

BACKGROUND AND PURPOSE: Ischemic cerebral injury is associated with activation of the blood coagulation cascade. To elucidate the contribution of fibrin formation to microvascular injury during focal cerebral ischemia and reperfusion, we have studied the time course and the localization of fibrin deposition in cerebral microvessels and the surrounding tissues during ischemia/reperfusion in a well-described nonhuman primate model. METHODS: Cerebral tissues from adolescent male baboons were examined after 2-hour middle cerebral artery occlusion (n = 3) and after 3 hours of middle cerebral artery occlusion and 1-hour (n = 6), 4-hour (n = 3), and 24-hour (n = 4) reperfusion; tissues from control primates (n = 3) also were examined. Fibrin deposition was detected by immunohistochemical techniques using the fibrin-specific monoclonal antibody MH-1. The number and size distribution of microvessels associated with fibrin were quantified by video-imaging microscopy. RESULTS: Fibrin was associated with microvessels only in the ischemic zone where severe neuronal injury was documented, its frequency increasing with the reperfusion period (F4,26 = 3.80, P < .05). Extravascular fibrin deposition was significantly increased by 24-hour reperfusion compared with the other subjects (P < .05). Preischemia infusion of the anti-tissue factor monoclonal antibody TF9-6B4 resulted in significant reduction of intramicrovascular fibrin (P < .038 versus no intervention) at 1-hour reperfusion but had no effect on extravascular fibrin deposition. CONCLUSIONS: These results suggest that microvascular fibrin deposition accumulates in a time-dependent manner during focal cerebral ischemia/reperfusion and that exposure of focal cerebral ischemia/reperfusion and that exposure of plasma to perivascular tissue factor is partially responsible for occlusion formation. During ischemia the large plasma protein fibrinogen extravasates and interacts with parenchymal tissue factor, forming significant extravascular fibrin by 24 hours of reperfusion.

Animals↗
PubMed1992

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Adhesion and spreading of cultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin beta chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B beta chain. After clotting with thrombin, this fibrin derivative lacking B beta 1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 15-42 of the fibrin beta chain.

Batroxobin↗
PubMed2003

Fibrin glue as a sealant for high-risk anastomosis in surgery for morbid obesity.

BACKGROUND: Fibrin sealants promote hemostasis and wound healing. Complex revisional surgery is performed for morbid obesity, and high-risk patients undergo weight loss surgery routinely. Fibrin sealant, Tisseel, was used by one surgeon on 120 consecutive patients at the gastrojejunal anastomosis in Roux-en-Y gastric bypass (RYGBP). We hypothesized that the application of fibrin sealant would decrease anastomotic leaks. METHODS: One surgeon (Surgeon A) used fibrin sealant for 120 consecutive patients, while two other surgeons (Surgeons B & C) served as controls and did not use fibrin glue for their last 120 patients. Surgeon A did not use fibrin glue in 120 patients to serve as an internal control. All 480 patients underwent a RYGBP. Fibrin glue was applied at the gastrojejunal anastomosis. RESULTS: The fibrin sealant group did not have any documented leaks on the previous 120 patients, while 5 patients with Surgeon B, 2 patients with Surgeon C and 1 patient with Surgeon A without fibrin sealant experienced enteric leaks requiring re-operation, drainage, or long-term total parenteral nutrition (N = 480 total patients). CONCLUSIONS: Fibrin sealant may be useful in preventing leaks and promoting healing of the "high risk" anastomosis during complex gastrointestinal surgery. While the cost of fibrin glue is to be considered, re-operation and management of subsequent enterocutanous fistulas or anastomotic strictures may be more costly than routine use for high-risk morbidly obese patients.

Adult↗
PubMed1999

Fibrinogen Niigata with impaired fibrin assembly: an inherited dysfibrinogen with a Bbeta Asn-160 to Ser substitution associated with extra glycosylation at Bbeta Asn-158.

A novel BbetaAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser-type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BbetaAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin gamma-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bbeta-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.

Amino Acid Substitution↗
PubMed2002

An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator.

During thrombolytic therapy and after recanalization is achieved, reduction in the volume of mural thrombi is desirable. Mural thrombi are known to contribute to rethrombosis and reocclusion. The lysis rate of mural thrombi has been demonstrated to increase with fluid flow in different experimental models, but the mechanisms responsible are unknown. An experimental and a theoretical study were developed to determine the contribution of outer convective transport to the lysis of mural fibrin clots. Normal human plasma containing recombinant tissue-type plasminogen activator (tPA; 0.5 microg/mL) was (re)perfused over mural fibrin clots with fluorescently labeled fibrin at low arterial, arterial, or higher wall shear stresses (4, 18, or 41 dyn/cm(2), respectively) and lysis was monitored in real time. Flow accelerated lysis, but significantly only at the highest shear stress: The average lysis front velocity was 3 x 10(-5) cm/s at 41 dyn/cm(2) vs. almost half of that at the lower shear stresses. Confocal microscopy showed fibrin fibers dissolving only in a narrow region close to the surface when permeation velocity was predicted to be low. A heterogeneous transport-reaction finite element model was used to describe mural fibrinolysis. After scaling the effects of outer and inner convection, inner diffusion, and chemical reactions, a simplified inner diffusion/reaction model was used. Correlation to fibrin lysis data in purified systems dictated higher rates of plasmin(ogen) and tPA adsorption onto fibrin and a decreased catalytic rate of plasmin-mediated fibrin degradation, compared with published parameters. At each shear stress, the model predicted a temporal pattern of lysis of mural fibrin (similar to that observed experimentally), and protease accumulation in a narrow fibrin region and significant lysis inhibition by plasma alpha(2)-antiplasmin (according to the literature). Increasing outer convection accelerated mural fibrinolysis, but the model did not predict the big increase in lysis rate at the highest shear stress. At higher than arterial flows, additional mechanisms not accounted for in the current model, such as fibrin collapse at the fibrin front, may regulate the lysis of mural clots and determine the outcome of thrombolytic therapy.

Blood Flow Velocity↗
PubMed2002

The use of fibrin sealant in urology.

PURPOSE: Fibrin sealant has been increasingly applied in various surgical fields, including urological surgery, in the last 2 decades. We determined the safety and efficacy of fibrin sealant in urological surgery and identified areas that need further clinical investigation. MATERIALS AND METHODS: A MEDLINE search of all available literature regarding the use of fibrin sealant was performed. All articles, including experimental animal studies, prospective and retrospective studies, case series and case reports of fibrin sealant for hemostasis and/or other urological applications, were identified and reviewed. RESULTS: Prospective randomized studies in the field of thoracic and trauma surgery show the efficacy and safety of fibrin sealant for hemostasis. Based on these data fibrin sealant has been used successfully for hemostasis during partial nephrectomy and traumatic renal reconstruction. A number of experimental animal studies, case series and case reports show the efficacy of fibrin sealant for ureteral anastomosis, microsurgical vasal anastomosis, fistula repair, circumcision and orchiopexy as well as it use as an adjunct in other areas of reconstruction. CONCLUSIONS: Fibrin sealant is an effective and safe topical agent for controlling surface bleeding during elective and trauma related urological procedures. Using fibrin sealant as an adhesive for reconstruction requires further prospective studies. The introduction of laparoscopic procedures in urology may expand the indications for fibrin sealant as an alternative method of tissue reapproximation. Limiting the routine use of fibrin sealant to procedures with demonstrable benefits is desirable and would lead to a cost saving approach.

Anastomosis, Surgical↗
PubMed2002

Immunoglobulin G from patients with antiphospholipid syndrome impairs the fibrin dissolution with plasmin.

Immunoglobulin G (IgG) isolated from normal human blood plasma stabilizes the structure of perfused crosslinked fibrin and prolongs the time for its dissolution with plasmin, when the fibrin surface is exposed to 500 s(-1) shear rate flow. The IgG from patients suffering in antiphospholipid syndrome with thrombotic complications exerts even stronger antifibrinolytic effect. A patient, whose IgG does not affect the fibrin dissolution with plasmin, displays a bleeding tendency. The shear stress-induced disassembly of the fibrin clots containing IgGs with antifibrinolytic potency occurs at a much more advanced stage of fibrin digestion, as evidenced by the electrophoretic pattern of the ureatreated samples. The antifibrinolytic effects are also produced under static conditions and these are caused by the variable portion of the IgG molecules (fragment Fab), whereas the constant part (fragment Fc) has no inhibitory effect. The IgGs with antifibrinolytic properties do not affect directly the plasmin activity in amidolytic assay, but the IgGs from APS patients obliterate the competition of the fibrin and the peptidyl-p-nitroanilide for the protease in the same assay system suggesting interference of the IgGs with the plasmin action on the fibrin substrate. Thus, the correlation of the clinical symptoms with the effect of the isolated IgG on the dissolution of perfused fibrin clots supports a physiological and a pathological role of IgG in the fibrinolytic process related to the variability of the cross-reactions of immunoglobulins with fibrin, fibrin degradation products or fibrin-plasmin complexes.

Adult↗
PubMed1990

Multimer size dependence of von Willebrand factor binding to crosslinked or noncrosslinked fibrin.

von Willebrand factor (vWF) is synthesized in endothelial cells (EC) and may be either secreted constitutively or stored in Weibel-Palade bodies (WPB) for regulated release. Because fibrin stimulates rapid vWF release from EC, we examined the binding of EC synthesized vWF to fibrin. Culture medium containing constitutively secreted vWF was removed from metabolically labeled primary cultures of human umbilical vein EC, and vWF released from WPB was obtained after stimulation by A23187. vWF-deficient fibrinogen with or without factor XIII was added to releasate or media and clotted with thrombin to form crosslinked or noncrosslinked fibrin. vWF was immunopurified from releasate or media before and after clotting, and the amount and multimeric pattern of vWF bound was determined after sodium dodecyl sulfate agarose gel electrophoresis. High molecular weight multimers of vWF, whether secreted constitutively or released from WPB, bound preferentially to fibrin. Multimers of greater than 20 subunits represented 60% +/- 4% (SEM) of A23187 released vWF and 11% +/- 5% of media vWF, but binding to fibrin was similar, 96% +/- 1% and 94% +/- 2%, respectively. A progressively smaller proportion of vWF bound as multimer size decreased, and dimeric vWF binding was least, with 34% +/- 5% binding from A23187 releasate and 51% +/- 4% from media. The amount of vWF binding to crosslinked or noncrosslinked fibrin was similar, and preferential binding of high molecular weight multimers occurred with both. As measured by enzyme-linked immunosorbent assay, 45% +/- 2% of constitutively secreted vWF bound to crosslinked fibrin and 50% +/- 2% to noncrosslinked fibrin. The propolypeptide of vWF did not bind to fibrin. These findings indicate that binding of EC secreted vWF binding to fibrin depends on multimeric size but not on factor XIII crosslinking. This suggests that vWF released from EC in the presence of fibrin will bind locally, thereby facilitating platelet adhesion to the hemostatic plug or thrombus.

Calcimycin↗
PubMed1990

Fibrin fragment D-dimer and fibrinogen B beta peptides in plasma as markers of clot lysis during thrombolytic therapy in acute myocardial infarction.

The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the "Thrombolysis in Myocardial Infarction II" (TIMI II) trial. Cross-linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1-42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15-42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1-42 in the fibrinopeptide beta 15-42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15-42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1-42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin-specific tag ELISA may provide more accurate information when correlated with clinical endpoints.

Antifibrinolytic Agents↗
PubMed1989

Fibronectin augments binding of fibrin to macrophages.

Because of the demonstrated ability of fibronectin to mediate particle uptake by macrophages and the demonstrated affinity of plasma fibronectin for fibrin, we investigated the ability of plasma fibronectin to augment macrophage binding of fibrin. Fibronectin significantly increased fibrin binding by elicited peritoneal macrophages and isolated hepatic Kupffer cells. The binding of fibrinogen was not augmented in the presence of fibronectin. The small amount of macrophage-associated fibrin observed in the absence of fibronectin was primarily internalized, whereas the increment in fibrin binding in the presence of fibronectin remained primarily surface bound, as indicated by susceptibility to removal by trypsin. An amino terminal fibrin-binding fragment of plasma fibronectin could similarly support binding of fibrin by peritoneal macrophages. Greater quantities of fibrin were associated with the macrophages in the presence of protease inhibitors, which inhibited elastase activity, but not in the presence of those that inhibited cathepsin activity, suggesting that an elastase-like protease may degrade surface-bound fibrin. Uptake of both fibrin and fibronectin was inhibited by prior treatment of cells with trypsin. Competitive binding studies suggested the presence of a high-affinity fibronectin receptor on peritoneal macrophages. Data from the current study thus support the conclusion that fibronectin augments binding of fibrin to the surface of mononuclear phagocytes.

Animals↗