Fibrinogen Function Research Articles

Fibrinogen facilitates the anti-tumor effect of nonnative endostatin

Endostatin is a potent inhibitor of tumor angiogenesis. Interestingly, nonnative endostatin also exhibits an anti-tumor effect, which remains a mystery so far. Here, we show that intravenous injection of nonnative endostatin results in tumor inhibition effect. Soluble and active endostatin is isolated from human blood after the addition of nonnative endostatin in vitro. By fractionation of the whole blood, we surprisingly identify fibrinogen specifically binding to and inhibiting the aggregation of nonnative endostatin. Moreover, the anti-tumor activity of nonnative endostatin is substantially impaired in fibrinogen-deficient mice. Our studies demonstrate that fibrinogen facilitates the anti-tumor effect of nonnative endostatin, which also provides new insights into the novel physiological function of fibrinogen.

Two cases of congenital dysfibrinogenemia associated with thrombosis – Fibrinogen Praha III and Fibrinogen Plzeň

Congenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous gamma Y363N mutation (Fibrinogen Praha III) and the heterozygous Aalpha N106D mutation (Fibrinogen Plzen), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzen fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzen. Praha III mutation was situated in the polymerisation pocket "a". The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzen mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector; and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.

Mechanisms Coupling Thrombin-Mediated Proteolysis to Metastasis.

Multiple lines of evidence point to a link between coagulation factors and malignancy. Numerous clinical studies have demonstrated a strong correlation between tumor cell-associated tissue factor (TF) expression and poor outcome for many types of cancer. While TF expression by tumor cells could promote metastasis through several mechanisms, comparative analyses of TF-sufficient and TF-deficient tumor cell lines in mice with and without key circulating hemostatic factors point to thrombin as the premier downstream effector coupling TF to metastasis. Furthermore, it appears that thrombin supports metastasis through mechanism(s) that are highly dependent on the availability of fibrinogen, factor XIII, and platelet function. The metastatic potential of TF-expressing tumor cells has been shown to be strongly diminished in mice with genetic defects in either fibrin formation or stabilization (e.g., Fib−/−, fXIII−/−), or platelet disorders (e.g., NF-E2−/−, Gαq−/−, PAR-4−/−). Interestingly, one mechanism through which tumor-cell-associated TF, prothrombin, and the platelet/fibrinogen axis all appear to support metastasis is by impeding natural killer (NK) cell-mediated clearance of newly formed micrometastases. While these results demonstrate a novel link between the hemostatic and innate immune systems, limiting NK cell function is clearly not the only mechanism coupling thrombin to tumor cell metastasis. Signaling through protease activated receptors (PARs) on cells other than platelets has been suggested to support tumor stroma formation and angiogenesis. Furthermore, thrombin-mediated signaling via PARs expressed by tumor cells themselves has been shown to support metastatic potential. Finally, thrombin may support metastatic potential by acting in concert on multiple substrates (e.g., platelet and endothelial cell-associated PARs, fibrinogen, fXIII, etc.) to promote the sustained, stable adherence of circulating tumor cells in distant vascular beds. In summary, thrombinmediated proteolysis appears to influence both extracellular and intracellular events vital to tumor cell dissemination. Therapeutic strategies designed to block tumor cell-associated thrombin generation/activity could prove effective in preventing or eliminating metastatic disease.

Laboratory testing for fibrinogen abnormalities

Fibrinogen is essential for the formation of a fibrin clot. Acquired and congenital disorders of fibrinogen may result in decreased concentration or altered function of fibrinogen, often leading to an increased risk of bleeding. Routine coagulation testing and specialized laboratory investigations can guide diagnosis in patients suspected of having a fibrinogen abnormality. This article summarizes the types of laboratory assays that are used to assess fibrinogen disorders, and key abnormalities found in different types of fibrinogen disorders.

Validation of thromboelastometry in horses

Thromboelastometry is used for identifying or monitoring coagulation abnormalities. It has been validated in several species but not in horses and the characteristics of the equine thromboelastogram have not yet been detailed. The purpose of this study was to validate a thromboelastometer to be used with equine blood and to define the normal equine thromboelastogram. A Rotem-gamma thromboelastometer (Pentapharm GmbH, Munich, Germany) was used on 38 citrated blood samples to investigate native coagulation, the intrinsic and extrinsic pathways, the function of fibrinogen (largely dependent on its concentration), and the presence of fibrinolysis. Using classic validation approaches, we evaluated the imprecision of the method and the influence of hemolysis and storage time and temperature. The normal thromboelastogram was defined in both saddle and racing horses (the latter sampled before and after the race). For imprecision tests, the analytical variations were <10%. The equine thromboelastogram had a pattern similar to those of other species, but the intrinsic and extrinsic pathways were less and more efficient, respectively. Reference intervals in racing horses, especially after exercise, were different from those of saddle horses, most likely due to a higher RBC mass. Coagulability decreased in hemolyzed samples and significant changes were found between nonrefrigerated and refrigerated blood samples stored for 20 hours. The Rotem-gamma thromboelastometer is a precise instrument for use with equine blood samples. The equine thromboelastogram is similar to that of other species, but reference intervals vary with aptitude and exercise. Hemolysis and refrigeration alter thromboelastometric results.

Alterations of Fibrinogen Structure in Human Disease

Products of normal and pathologic metabolism can react with proteins to cause covalent modification. When such modifications affect fibrinogen they can potentially alter fibrinogen function. Those that have been best studied are oxidation, nitration, homocysteinylation and glycation. It appears that the clottability of fibrinogen is maintained unless the degree of modification is extensive. However, modest degrees of fibrinogen modification can alter the rate of assembly of fibrin monomers into a fibrin clot and the fiber structure and packing. In addition, some types of modification affect lysine residues that are critical to binding, activation and activity of fibrinolytic enzymes. Any of these alterations could potentially affect the susceptibility of fibrin clots to fibrinolysis, and have been shown to do so in vitro. In the case of homocysteinylation and glycation, good evidence exists that fibrinogen modification affects clot stability in vivo. However, direct evidence is still lacking that these modifications contribute to the increased atherothrombotic risk associated with hyperhomocysteinemia and diabetes.

Influence of direct thrombin inhibitor argatroban on coagulation assays in healthy individuals, patients under oral anticoagulation therapy and patients with liver dysfunction

We assumed that argatroban, a direct thrombin inhibitor, has a strong influence on different coagulation tests which is even more pronounced in patients with an established reduced factor activity like those under oral anticoagulation therapy or with liver dysfunction. To validate this influence we spiked plasma samples from healthy individuals, patients under oral anticoagulation therapy or with liver dysfunction with increasing argatroban concentrations (0-2000 ng/ml) and performed routine laboratory coagulation tests. Consequently, prothrombin time, activated partial thromboplastin time, thrombin time, batroxobin time, coagulation factor activity (FII-FXIII), protein S (activity), protein C (chromogen) and fibrinogen (derived and Clauss fibrinogen method) were measured. Furthermore, the influence of argatroban on the induced platelet aggregation was evaluated. Argatroban interference on standard coagulation assays differed markedly depending on the different subgroups of patients investigated. Prolongation of prothrombin time by argatroban (at 2000 ng/ml 2.7-fold in healthy persons) was significantly higher in oral anticoagulation therapy (3.9-fold) and even more pronounced in liver dysfunction (6.0-fold). The fibrinogen concentration was determined falsely even at low-argatroban concentrations using functional methods in healthy persons and all patient subgroups. The influence of argatroban on standard laboratory coagulation tests is significantly increased by a preexisting factor deficiency. Functional fibrinogen measurement may be helpful to assess in-vivo fibrinogen function but should be avoided to evaluate fibrinogen concentration in argatroban treated patients. Argatroban had no influence on chromogenic protein C measurement, batroxobin time and induced platelet aggregation. Knowledge of argatroban interference is a prerequisite for the reliable interpretation of coagulation assays.

Abnormalities in gγ region of fibrinogen molecule and influences in assembly, secretion and function of intact fibrinogen

Abnormalities in gγ region of fibrinogen molecule and influences in assembly, secretion and function of intact fibrinogen

Citrate Anticoagulant Artificially Masks the Hemostatic Effect of Recombinant Factor VIIA in Dilutional Coagulopathy.

Introduction: Dilutional coagulopathy may develop in massively bleeding patients who are substituted with synthetic colloid plasma expanders. A series of recent studies have demonstrated that synthetic colloids may induce abnormal function of fibrinogen by compromising fibrin polymerization and substitution with a fibrinogen concentrate appeared to reverse this coagulopathy and arrest traumatic bleeds. Clinical experiences, several case reports as well as a randomised trial point toward a beneficial effect of rFVIIa in control of massive bleeding associated with hemodilution caused by excessive volume substitution. Our haemostasis center recently put forward that rFVIIa, in vitro, was unsuccessful in correction of dilutional coagulopathy induced by HES 130/04. In addition, other investigations utilising a rabbit model have indicated that colloid hemodilution may attenuate the haemostatic potential of rFVIIa. For half a Century laboratory coagulation tests have been carried out using re-calcified citrated blood. However, recent experimental work has revealed that citrate dependent calcium chelation significantly changes the dynamic course of thrombin generation, suspectedly due to interference with the enzymatic properties of coagulation factors. In addition it has been documented that citrate and calcium chelation interferes with the metabolism in platelets. In the present study we aimed at investigating the haemostatic effect of rFVIIa in a laboratory whole blood model of colloid hemodilution using citrate as well as iso-citrate stabilized blood comparing to native whole blood and whole blood stabilized with corn trypsin inhibitor (CTI) that specifically blocks factor XIIa.Materials and Methods: Following informed consent 11 healthy male volunteers with a mean age of 30 years (range 26–38 years) delivered blood for study. Dynamic whole blood coagulation profiles were recorded using thrombelastography activated with minute amounts of tissue factor in a model of ex vivo hemodilution with HES 130/0.4 in a prospective approach. Analyses were evaluated at 30% dilution level, and following ex vivo addition of rFVIIa to whole blood collected into tubes containing citrate, iso-citrate, CTI, or no stabilizer.Results: Hemodilution with HES 130/0.4 induces a coagulopathy characterized by a reduced maximum rate of clot formation and a pronounced reduction in the final clot firmness. With all test mediums investigated, rFVIIa significantly shortened the clot initiation phase. In cases of native whole blood and CTI stabilized whole blood rFVIIa shortens the clotting time but also demonstrated an acceleration of the maximum velocity of clot formation.Conclusion: When citrate or iso-citrate is used as anticoagulants in thromboelastographic clotting assays, these anticoagulants may artificially mask the haemostatic effect of rFVIIa in colloid hemodilution blood. The effect in vitro of rFVIIa in citrated blood samples may significantly underestimate the haemostatic potential of rFVIIa. In cases where the hemostatic potential of rFVIIa is tested in vitro a potentially efficacious rescue treatment may be delayed or excluded.

Acquired dysfibrinogenemia caused by monoclonal production of immunoglobulin light chain

Disorders of fibrinogen are usually caused by genetic mutations that result in low protein levels (hypofibrinogenemia) or an abnormal molecule (dysfibrinogenemia). However, environmental and plasma factors can have an acquired effect on its expression or function. For example, antibodies can bind fibrinogen and/or fibrin to interfere with polymerization and inhibit coagulation. The objective here was to determine the cause of dysfibrinogenemia in a 63-year-old man. Despite a low functional fibrinogen concentration and prolonged thrombin time, no inherited fibrinogen abnormality could be detected after extensive protein analysis and gene sequencing. Thus, electrophoresis methods and fibrinogen binding studies were used to establish the cause of the acquired dysfibrinogenemia. An immunoglobulin lambda light chain was found to bind fibrinogen as a monomer. It had no significant effect on fibrinopeptide release, but caused substantial defects in all other stages of thrombin-catalyzed fibrin polymerization. Binding to fibrinogen also seemed to prevent the light chain from being filtered through the kidneys, causing only low levels of it in the urine. Once in the urine, the lambda chain lost its anti-fibrinogen activity, apparently due to dimerization. The 63-year-old patient acquired dysfibrinogenemia from a monoclonal production of lambda light chain that bound and inhibited the function of fibrinogen. At age 64.5 he was diagnosed with monoclonal gammopathy of undetermined significance, explaining the abnormal immunoglobulin chain production. This case was particularly unusual in that the inhibition of fibrin polymerization was caused by a single immunoglobulin light chain, rather than by a whole antibody molecule.

The role of ascorbate and histidine in fibrinogen protection against changes following exposure to a sterilizing dose of γ-irradiation

Sodium ascorbate and histidine were employed to protect fibrinogen against modifications followed by a gamma-irradiation process that could potentially inactivate the blood-borne viruses in plasma-derived products. Fibrinogen was irradiated (50 kGy total dose, on dry ice) using a 60Co source. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Carbonyl groups were measured by the 2,4-dinitrophenylhydrazine-coupled method, and the fibrinogen clotting activity was assessed by different functional assays. In irradiated fibrinogen, the carbonyl group concentration was elevated three-fold versus control; and moderate fragmentation of largely Aalpha and Bbeta chains was revealed. The rate of thrombin-catalyzed fibrinogen polymerization was inhibited (average 50%) with normal fibrinopeptide release and with a minor decrease of total clottable fibrinogen and alpha-polymer formation. Ascorbate reduced the incorporation of carbonyls to the fibrinogen molecule (by > 50% at 50 mmol/l; P < 0.001). Contrary to ascorbate, which alone delayed the fibrinogen polymerization rate, histidine abolished irradiation-induced inhibition of fibrinogen polymerization (by 80% at 50 mmol/l; P < 0.001). In conclusion, even though ascorbate effectively protects fibrinogen from oxidation due to its adverse effects on fibrinogen function, it may not serve as a suitable radioprotective. On the contrary, the first definite evidence is provided that radiation-sterilized fibrinogen in the presence of histidine greatly retains its clotting capability.

Analysis of hemostasis alterations in sepsis

This laboratory study tested new methods to analyze hemostasis alterations in septic patients. Samples of ethylenediamine tetraacetic acid (EDTA) plasma and citrated plasma were collected from 62 patients with clinical diagnosis of sepsis. Additionally, a subset of EDTA-plasma samples from each patient was stabilized 1 + 1 with 2.5 mol/l arginine, pH 8.6, to conserve the real hemostasis activation state. EDTA-arginine plasma, EDTA plasma and citrated plasma samples were tested in duplicate. The patients at admission to the intensive care unit had 36 +/- 26 (normal, 0.8 +/- 0.2) ng/ml global endotoxin reactivity, 188 +/- 66% (normal, 100 +/- 20%) fibrinogen function, 179 +/- 66% (normal, 100 +/- 20%) fibrinogen antigen, 4.0 +/- 3.6 (normal, 0.049 +/- 0.025) microg/ml D-dimer, 313 +/- 307% (normal, 100 +/- 30%) plasmin-antiplasmin complex, 8.7 +/- 11.4 (normal, 1.1 +/- 0.7) U/ml plasminogen activator inhibitor-1, 12.1 +/- 10.5 (normal, 1.3 +/- 0.4) ng/ml thrombin-antithrombin III complex, 173 +/- 62% (normal, 100 +/- 20%) thrombin, 568 +/- 225 (normal, 140 +/- 42) pg/ml tissue factor, and 2.56 +/- 2.48 (normal, 0.19 +/- 0.04) microg/ml soluble intercellular adhesion molecule-1. Endotoxin (lipopolysaccharide and/or beta-glucan) reactivity (EDTA plasma), fibrinogen function + antigen + ratio and plasminogen activator inhibitor-1 (citrated plasma), and D-dimer, soluble intercellular adhesion molecule-1, thrombin activity (EDTA-arginine-stabilized plasma) presented large aberrations in septic patients when compared with normal values and may therefore be particularly interesting as markers of hemostasis alteration. Whether the observed alterations are of clinical significance has to be determined in well defined patient groups.

Hematopoietic stem cell transplantation for Hodgkin’s disease in a patient with dysfibrinogenemia and thrombosis

Dysfibrinogenemia is a disorder of fibrinogen structure and is associated with a functional abnormality. Since fibrinogen is a key component of both the procoagulant and fibrinolytic pathways, defects in fibrinogen function can be associated with increased risk for both hemorrhage and thrombosis. Management of patients with dysfibrinogenemia and a thrombotic tendency usually involves long-term anticoagulation. A 36-year-old male with relapsed nodular sclerosing Hodgkin's was found to have a prolonged prothrombin time, low fibrinogen activity and a normal fibrinogen antigen during evaluation for a hematopoietic peripheral blood stem cell transplant. His past medical history was significant for an acute myocardial infarction and two episodes of acute pancreatitis. His father had dysfibrinogenemia complicated by multiple thrombotic episodes. A trans-esophageal echocardiogram revealed two thrombi, one each in the superior vena cava and the descending aorta. He was treated with enoxaparin and received peripheral blood stem cell transplantation. An effort was made to maintain his fibrinogen activity levels at 200 mg/dL using cryoprecipitate. A month following the transplant he developed a new thrombus in the right internal jugular vein, while on enoxaparin and he was started on argatroban and cryoprecipitate followed by fondaparinux. A repeat echocardiogram six weeks later demonstrated that the burden of thrombus both in the right atrium and descending aorta was significantly lower. This is the first case report of a patient with dysfibrinogenemia undergoing peripheral blood stem cell transplantation. Conventional anticoagulant therapy and cryoprecipitate seem to be a reasonable management strategy to prevent thrombosis in a patient with dysfibrinogenemia and a thrombophilic tendency. Secondly, fondaparinux can be used in cases of failure of therapy with low molecular weight heparins and may actually be superior to low molecular weight heparins, especially in patients with dysfibrinogenemia.

Hepatocyte-derived fibrinogen-related protein-1 is associated with the fibrin matrix of a plasma clot

In order to study the multiple functions of fibrinogen and fibrin, we are investigating which proteins bind to the fibrin matrix of a plasma clot by using a proteomic approach. Extracts from washed plasma clots were analysed by 2-D gel electrophoresis. A relatively abundant spot was identified as hepatocyte-derived fibrinogen-related protein-1 (HFREP-1) by MALDI-TOF analysis, molecular mass (34 kDa), iso-electric point (p I 5.5) as well as by Western blot analysis. HFREP-1 in plasma almost completely bound to the fibrin matrix during clot formation. Several purified fibrinogen preparations proved to be contaminated with HFREP-1. It is concluded that HFREP-1 (also named hepassocin), a protein with liver cell growth regulatory properties, occurs in plasma and strongly associates with fibrin and possibly fibrinogen.

Fibrinogen Seoul (FGG Ala341Asp): A Novel Mutation Associated With Hypodysfibrinogenemia

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in fibrinogen function. The molecular basis of hypodysfibrinogenemia was investigated in a 66-year-old woman with peripheral artery obstructive disease and in her family members. Plasma level of functional fibrinogen determined using the Clauss method was lower (75 mg/dL; normal, 140-460 mg/dL) than that measured with immunologic nephelometric assay (137 mg/dL; normal, 180-400 mg/dL). Similar results were also observed in two family members through two generations. DNA was extracted from whole blood, and the coding regions and intron/exon boundaries of gamma chain gene (FGG) were amplified. A novel (Fibrinogen Seoul) heterozygous FGG mutation (GCT->GAT, Ala341Asp) was identified in all three affected family members. Thrombin-catalyzed polymerization was found to be defective on the analysis of purified fibinogen from the propositus. Molecular modeling also showed a conformational change of fibrinogen structure.

Functional analysis of recombinant Bβ15C and Bβ15A fibrinogens demonstrates that Bβ15G residue plays important roles in FPB release and in lateral aggregation of protofibrils

Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant-variant fibrinogens. We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala. Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys fibrinogen, functional analysis indicated (a) no thrombin-catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin- and reptilase-catalyzed fibrin polymerizations. For Bbeta15Ala fibrinogen, such analysis indicated slight impairments of both thrombin-catalyzed FPB release and lateral aggregation in thrombin-catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase-catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). We conclude that a region adjacent to Bbeta15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.

Modifications of flow measurement to determine fibrin gel permeability and the preliminary use in research and clinical materials

Our earlier investigations employing a flow measurement have yielded intriguing findings as to what governs the fibrin network porosity. To make the method suitable for use by more groups with various laboratory conditions, sample materials or study purposes, we simplified the essential equipment and thereby minimized the sample volume to 250 microl in comparison with the need for 3000 microl in the previous method. To assess whether the fibrin gel permeability depends on changes in thrombin generation potential and/or fibrinogen clotting property, different concentrations of thrombin with or without frozen-thawed platelets, serving as phospholipids, were used. The platelets and 0.05 IU/ml thrombin were added to plasma samples from patients with previous myocardial infarction. The fibrin gel permeability, expressed as Darcy constant (Ks), was decreased compared with that in controls, supporting findings about high risk of thromboembolism in this disease due to increases of thrombin activity and fibrinogen function. When 0.4 IU/ml thrombin was used in samples provided by 10 healthy individuals treated with acetysalicylic acid, Ks levels were increased during versus before therapy. Since almost no thrombin generation was found in the samples with the higher dose of exogenous thrombin, we considered that modifications in fibrinogen clotting property by acetysalicylic acid rendered the fibrin network more permeable. In summary, as the reproducibility remains satisfactory (coefficient of variation < 10%) despite aforementioned modifications in the equipment and reagents, any interested laboratory ought to be able to repeat the method. Assays of fibrin permeability in such a simple way may help to determine the fibrin clot stability in pathological/pharmacological studies, and probably serve as a tool to estimate thromboembolism risk in clinical materials, such as patients with cardiovascular diseases.

ClfA221–550, a fibrinogen-binding segment of Staphylococcus aureus clumping factor A, disrupts fibrinogen function

Clumping factor A (ClfA) is a surface protein of Staphylococcus aureus bacteria known for its ability to bind the C-terminus of plasma fibrinogen gamma chain, which participates in mediating fibrinogen-platelet interaction and fibrin cross-linking, resulting in thrombus formation. With an aim to develop agents that block fibrinogen gamma chain C-terminus, the fibrinogen-binding segment of ClfA locating at residues 221-550 was produced by recombinant technology and tested for its ability to inhibit platelet functions and fibrin clot formation. Recombinant ClfA(221-550) bound fibrinogen and blocked fibrinogen-platelet interaction, resulting in the inhibition of both ADP- and collagen-induced platelet aggregations. ClfA(221-550) also affected fibrin clot formation, in which factor XIIIa-mediated cross-linking of fibrinogen gamma chains was abrogated by ClfA(221-550) leaving the release of fibrinopeptides A and B from fibrinogen by thrombin unaltered, indicating that ClfA(221-550) interfered with fibrin clot formation without affecting thrombin's catalytic activity. Platelet-mediated clot retraction depends on both platelet-fibrinogen interaction and fibrin clot formation, which makes platelet thrombus less susceptible to fibrinolysis. At the concentration that reduced platelet aggregation by 40%, ClfA(221-550) prevented platelet-mediated clot retraction, whereas the glycoprotein IIb/IIIa antagonist tirofiban needed a higher concentration in inhibiting clot retraction than inhibiting platelet aggregation. By virtue of the multiple effects of ClfA(221-550) on platelet aggregation, fibrin clot formation and platelet-mediated clot retraction, the binding of ClfA(221-550) to fibrinogen merits further investigation for its potential as a new antithrombotic agent.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Peroxynitrite decreases hemostasis in human plasma in vitro.

Coagulopathy has been associated with clinical scenarios that involve reactive nitrogen species such as peroxynitrite (OONO-). Further, OONO- decreases tissue factor and fibrinogen function in vitro. Thus, we hypothesized that exposure of plasma to the OONO- generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide, would result in a decrease in hemostatic function via diminished coagulation protein activity. Hemostatic function of plasma exposed to SIN-1 (0, 1, 5, and 10 mM for 60 min at 37 degrees C) was assessed with thrombelastography, activated partial thromboplastin time, and prothrombin time in the presence or absence of superoxide dismutase (SOD) or an OONO- scavenger. SIN-1 exposure resulted in a significant (P < 0.05), dose-dependent decrease in plasma hemostatic function and concurrent significant (P < 0.05) decreases in activities of factor VII, factor VIII complex, and factor X. Fibrinogen concentration was not affected by SIN-1. Antithrombin and protein C activity also decreased significantly (P < 0.05). Coincubation with SOD or an OONO- scavenger significantly (P < 0.05) attenuated SIN-1 mediated changes in hemostasis and procoagulant/ anticoagulant activity. We conclude that OONO- may decrease hemostatic function in human plasma by nitration of key procoagulants and that OONO- may play a significant role in hemorrhagic states.