fibrin and glycyl-prolyl-arginyl-proline

The role of platelet activation in clinical settings is controversial, partly because the methods used to detect platelet activation (e.g. platelet aggregation and radioimmunoassays of plasma beta-thromboglobulin and platelet factor 4) are plagued by major methodological problems. Clinical studies that utilize flow cytometric assays of washed platelets are also susceptible to artefactual in vitro platelet activation. Whole blood flow cytometry circumvents many of these methodological problems. However, a major limitation in previously described whole blood flow cytometric assays is their inability to study the effect on platelet activation of thrombin, the most important platelet agonist in vivo. This article reviews the method and clinical applications of a new flow cytometric assay in which platelet activation by thrombin is directly measured in whole blood through the use of the peptide Gly-Pro-Arg-Pro (GPRP). GPRP inhibits thrombin-induced fibrin clot formation and platelet aggregation, but not thrombin-induced platelet activation. The presently described assay of platelet activation by thrombin in the physiological milieu of whole blood should be widely applicable to the many clinical settings (e.g. arterial thrombosis) in which platelet activation by thrombin is postulated to play an important role.

Topics: Amino Acid Sequence; Exercise; Fibrin; Flow Cytometry; Humans; Molecular Sequence Data; Oligopeptides; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Thrombin

Thrombin activates fibrinogen and binds the fibrin E-domain (K. A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (T. Large increases in T. TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin.. The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.

Topics: Amino Acid Chloromethyl Ketones; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinogens, Abnormal; Humans; Oligopeptides; Protein Stability; Thrombin; Transition Temperature

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Topics: Adult; Aged; Blood Loss, Surgical; Blood Viscosity; Cardiac Surgical Procedures; Dose-Response Relationship, Drug; Equipment Design; Female; Fibrin; Healthy Volunteers; Hemorheology; Humans; In Vitro Techniques; Male; Middle Aged; Oligopeptides; Predictive Value of Tests; Protein Multimerization; Thoracic Surgical Procedures; Thrombelastography; Thrombin; Tissue Plasminogen Activator

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding "cap." These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

Topics: Blood Platelets; Crotalid Venoms; Fibrin; Fibrinolysin; Fibrinolysis; Fibrinolytic Agents; Flow Cytometry; Hemostatics; Hirudins; Lectins, C-Type; Microscopy, Confocal; Oligopeptides; Phosphatidylserines; Protein Binding; Rheology; Shear Strength; Thrombin; Thrombosis; Tirofiban; Tissue Plasminogen Activator; Tyrosine; Urokinase-Type Plasminogen Activator

Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbβ3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.

Topics: Blood Coagulation; Blood Platelets; Blotting, Western; Female; Fibrin; Fibrinogen; Flow Cytometry; Humans; Microscopy, Confocal; Oligopeptides; Phosphatidylserines; Platelet Aggregation; Polymerization; Thrombasthenia; Thrombosis; Thrombospondins; Transglutaminases

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β₃ antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Topics: Blood Coagulation; Blood Platelets; Cytochalasin B; Dipeptides; Fibrin; Humans; Microscopy, Confocal; Oligopeptides; Phosphatidylserines; Platelet Glycoprotein GPIIb-IIIa Complex; Thrombin

At sites of vascular injury, thrombin is an important mediator in thrombus growth and stability. Using microfluidic flow devices as well as patterned surfaces of collagen and tissue factor (TF), we sought to determine the role that fibrin plays in clot stability without interfering with the production of thrombin.. We deployed an 8-channel microfluidic device to study coagulation during corn trypsin inhibitor-treated (XIIa-inhibited) whole blood perfusion over lipidated TF linked to a fibrillar collagen type 1 surface. Clot growth and embolization were measured at initial inlet venous (200 s(-1)) or arterial (1000 s(-1)) wall shear rates under constant flow rate or pressure relief mode in the presence or absence of Gly-Pro-Arg-Pro (GPRP) to block fibrin polymerization. Numerical calculations for each mode defined hemodynamic forces on the growing thrombi. In either mode at inlet venous flow, increasing amounts of TF on the surface led to a modest dose-dependent increase (up to 2-fold) in platelet deposition, but resulted in massive fibrin accumulation (>50-fold) only when exceeding a critical TF threshold. At a venous inlet flow, GPRP led to a slight 20% increase in platelet accumulation (P<0.01) in pressure relief mode with thrombi resisting ≈1500 s(-1) before full channel occlusion. GPRP-treated thrombi were unstable under constant flow rate, where shear forces caused embolization at a maximum shear rate of ≈2300 s(-1) (69 dynes/cm2). In constant flow rate mode, the nonocclusive platelet-fibrin deposits (no GPRP) withstood maximum shear rates of ≈29 000 s(-1) (870 dyne/cm2) at ≈95% of full channel occlusion. For arterial inlet shear rate, embolization was marked for either mode with GPRP present when shear forces reached 87 dynes/cm2 (≈2900 s(-1)). Under constant flow rate, platelet-fibrin deposits (no GPRP) withstood maximums of 2400 dynes/cm2 (80,000 s(-1)) at ≈90% of full channel occlusion prior to embolization.. Fibrin increased clot strength by 12- to 28-fold. Under pressure relief mode, ≈2-fold more fibrin was produced under venous flow (P<0.001). These studies define embolization criteria for clots formed with surface TF-triggered thrombin production (±fibrin) under venous and arterial flows.

Topics: Adult; Anticoagulants; Blood Coagulation; Blood Platelets; Collagen Type I; Computer Simulation; Embolism; Factor XIIa; Fibrin; Humans; Liposomes; Male; Microfluidic Analytical Techniques; Microscopy, Confocal; Models, Biological; Numerical Analysis, Computer-Assisted; Oligopeptides; Plant Proteins; Platelet Adhesiveness; Platelet Aggregation; Regional Blood Flow; Stress, Mechanical; Thrombin; Thromboplastin; Thrombosis; Time Factors; Young Adult

The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the gamma- and beta-modules. Of the potential knob-hole interactions--A:a, B:b, A:b, and B:a--the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers-based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the gammaD364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (beta15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the beta15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.

Topics: Amino Acid Motifs; Amino Acid Substitution; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Models, Molecular; Mutation, Missense; Oligopeptides; Optical Tweezers; Peptide Fragments; Point Mutation; Protein Binding; Protein Conformation; Protein Interaction Mapping; Protein Structure, Tertiary; Recombinant Fusion Proteins; Thrombin

D-dimer of human fibrin was used as antigen to obtain monoclonal antibodies (mAbs). We have obtained 16 hybridomas producing mAbs of different specificity. Only two of these mAbs inhibited fibrin polymerization. They are of the IgG-class. One mAb (II-4d) inhibited fibrin polymerization to 100% and another (II-3b) to 60% at a molar ratio mAb/fibrin=1.0. Fab-fragments of these mAbs inhibited fibrin polymerization completely at the same molar ratio. The epitopes for the mAbs studied are situated in the NH2-terminal part of the gamma-chain in fibrin D-domain. Electron microscopy showed that fibrin was in monomeric form in the presence of these mAbs or their Fab-fragments. Thus, these mAbs stop the initial step of fibrin polymerization, i.e. protofibril formation. Only one site of protofibril formation is known now in COOH-terminal half of the D-domain gamma chain named "a" site, which is complementary to the "A" site in the central E-domain of fibrin molecule. Our experiment with immobilized GPRP showed that the "a" site in fibrin D-fragment preserved its binding activity to GPRP when the D-fragment was complexed with mAbs-inhibitors of fibrin polymerization. Thus, these two mAbs inhibit fibrin polymerization not by blocking the sites "a" but either by blocking another (not "a") specific site in D-domain or by steric hindrance of highly organized fibrin polymerization process.

Topics: Antibodies, Monoclonal; Antibody Specificity; Biopolymers; Blood Coagulation; Enzyme-Linked Immunosorbent Assay; Epitopes; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Humans; Hybridomas; Immunoglobulin Fab Fragments; Oligopeptides; Protein Structure, Tertiary

Infective endocarditis (IE) caused by Staphylococcus aureus is associated with significant morbidity and mortality rates. Platelets play a dual role as adhesive cells forming associates with bacteria as well as specialized inflammatory cells. The specific role of the various factors involved in bacteria-platelet association has not yet been fully elucidated.. We observed a dramatic increase in the capability to bind S aureus when platelets were activated with thrombin (from 5% to 30%, P<0.001). To pinpoint platelet-binding sites involved in the interaction, platelets from knockout mice and from patients with selective inherited deficiency of membrane proteins or of granules were used. CD36, GPIIb/IIIa, and P-selectin were excluded as receptors for S aureus. Platelets from patients with alpha-delta-storage pool disease and Gray platelet syndrome indicate the requirement of alpha-granule contents. Platelet activation by ADP did not promote platelet-S aureus associate formation, although these platelets were covered with bound fibrinogen. Only small numbers of associates between fibrinogen-covered bacteria and ADP-activated platelets were observed. Formation of fibrin alone was also not sufficient to induce association. Only when fibrin formation and platelet activation occurred together were large numbers of associates formed (P<0.001). A potential receptor for fibrin on S aureus is clumping factor A. Addition of thrombospondin-1 to control platelets increased the number of associates (P=0.02).. Soluble fibrin but not fibrinogen is the main mediator of platelet-S aureus association. In addition, platelet activation and the release of alpha-granule contents, particularly thrombospondin-1, is a requirement for platelet-S aureus association.

Topics: Adenosine Diphosphate; Ancrod; Animals; Bacterial Adhesion; Blood Platelet Disorders; Blood Platelets; Coagulase; Cytoplasmic Granules; Fibrin; Fibrinogen; Hirudins; Humans; Mice; Mice, Knockout; Oligopeptides; Platelet Activation; Protein Binding; Staphylococcus aureus; Thrombin; Thrombospondin 1

The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its alpha chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIIIa-catalyzed end-to-end ligation of the gamma chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIIIa reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of alpha chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.

Topics: Cross-Linking Reagents; Fibrin; Humans; Oligopeptides; Transglutaminases

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the alpha chain of one fibrin molecule and the C-terminal region of a gamma chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) gamma chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the alpha chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

Topics: Dimerization; Fibrin; Models, Molecular; Molecular Sequence Data; Oligopeptides; Protein Conformation

Topics: Amino Acid Sequence; Blood Platelets; Cell Membrane; Enzyme Activation; Factor XI; Feedback; Fibrin; Humans; Molecular Sequence Data; Oligopeptides; Plasma; Thrombin

A chimeric plasminogen activator, GPRP-u-PA (144-411), consisting of the Gly-Pro-Arg-Pro tetrapeptide fused to the N-terminal of a truncated urokinase-type plasminogen activator (comprising Leu 144 through Leu 411), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. After renaturation, the chimera was purified to homogeneity with specific amidolytic activity of 100,000 IU/mg protein. The chimera showed 6-fold greater affinity for fibrin clots than native low molecular weight urokinase (LUK) and 1.5-fold greater affinity than a chemical conjugate, GPRP-LUK, generating via coupling Gly-Pro-Arg-Pro tetrapeptide to native low molecular weight urokinase. The chimera had 2 to 3 fold greater fibrinolytic potency than native LUK in vitro. Fibrinogen had no influence on fibrinolysis of the chimera. The chimera consumed much less fibrinogen than native LUK.

Topics: Base Sequence; Blotting, Western; Cloning, Molecular; Escherichia coli; Fibrin; Fibrinogen; Fibrinolysis; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Oligodeoxyribonucleotides; Oligopeptides; Recombinant Fusion Proteins; Urokinase-Type Plasminogen Activator

Two low molecular weight urokinase derivatives were obtained by covalent coupling of a synthetic Gly-Pro-Arg-Pro tetrapeptide to peptide A of low molecular weight urokinase and exchanging the native peptide A of low molecular weight urokinase with Gly-Pro-Arg-Pro-peptide A to obtain derivative I or direct conjugation of Gly-Pro-Arg-Pro tetrapeptide to low molecular weight urokinase to obtain derivative II. In caseinolytic assay, fibrin can stimulate the two derivatives to activate plasminogen. But the two derivatives showed different kinetic behaviors. The derivative I displayed immediate onset of lysis and derivative II displayed a lag phase.

Topics: Caseins; Enzyme Activation; Fibrin; Fibrinolysis; Kinetics; Oligopeptides; Plasminogen; Protein Conformation; Urokinase-Type Plasminogen Activator

A Gly-Pro-Arg-Pro tetrapeptide, homologous to amino-terminal segment of the human fibrin alpha chain after the release of the fibrinopeptide A, was covalently coupled to peptide A of low molecular weight urokinase. The resulting derivative gained increased affinity for fibrin. In caseinolytic assay, fibrin can stimulate the derivative to activate plasminogen. The derivative had two-fold greater fibrinolytic potency than native low molecular weight urokinase and its affinity for fibrin clot was 3.9-fold higher than that of low molecular weight urokinase.

Topics: Chromatography, Affinity; Fibrin; Fibrinolysis; Humans; Molecular Weight; Oligopeptides; Urokinase-Type Plasminogen Activator

Fibrinogen labeled with fluorescein isothiocyanate (FITC) was tested for its ability to serve as a template for macromolecular assembly as well as to provide a fluorogenic signal to allow continuous monitoring of plasminogen activation and fibrinolysis. As dilute solutions of FITC-fibrinogen or FITC-fibrin fiber suspension were degraded during lysis, release of fluorescent fragments abolished proximity-based quenching and resulted in a 2.0- or 3.6-fold increase in fluorescence intensity, respectively. Addition of plasmin at a final concentration of 10 pM to FITC-fibrinogen (10 nM) produced a detectable level of fluorescence dequenching. The assay had sufficient sensitivity to detect plasmin activity in the presence of excess antiplasmin activity, indicating the dissociation of a reversible antiplasmin-plasmin complex. The detection limit of the reaction assay was 20 pM and 200 pM of recombinant tPA and urokinase, using 10 nM FITC-fibrin and 10 nM and 5 nM plasminogen, respectively. The 10-fold greater sensitivity of the assay for tPA was likely due to the molecular assembly of tPA and plasminogen on the FITC-fibrin. Addition of thrombin (1 U/ml) and plasmin (0.1 nM) to 10 nM FITC-fibrinogen produced fluorescence quenching at first due to fibrinogen polymerization followed by dequenching due to fibrinolysis. Addition of 10 mM epsilon-aminocaproic acid to mixtures of thrombin and plasmin allowed the quenching assay of thrombin activity in the presence of active plasmin. FITC-fibrinogen could be copolymerized with recalcified platelet poor plasma (isolated from citrated whole blood) to yield fibrin that was fluorogenic. Dequenching was observed when plasmin was used to degrade the fibrin formed from the platelet poor plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-2-Antiplasmin; Amino Acid Sequence; Antifibrinolytic Agents; Blood Proteins; Enzyme Activation; Fibrin; Fibrinogen; Fibrinolysin; Fibrinolysis; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fluorometry; Humans; Macromolecular Substances; Molecular Sequence Data; Oligopeptides; Plasminogen; Protein Binding; Recombinant Proteins; Sensitivity and Specificity; Solutions; Surface Properties; Templates, Genetic; Thrombin; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator

Ancrod, a thrombin-like enzyme, has been used as defibrinogenating agent in prevention of venous thrombosis. This study showed ancrod in citrated plasma elevated 6-keto PGF1 alpha production of human umbilical vein endothelial cells; this increment of 6-keto PGF1 alpha was completely inhibited by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The enhanced prostacyclin production, but not basal level of prostacyclin, was inhibited by actinomycin D and cycloheximide. In washed aspirin-pretreated cells, ancrod-formed fibrin induced restoration of prostacyclin production by a cycloheximide- and actinomycin D-sensitive process. Ancrod-formed fibrin stimulated synthesis of cyclooxygenase as probed by Western blotting and this enhancement was blocked by actinomycin D and cycloheximide. In conclusion, we first report that ancrod-formed fibrin stimulates prostacyclin production of human endothelial cells and this event is dependent on de novo synthesis of cyclooxygenase.

Topics: 6-Ketoprostaglandin F1 alpha; Ancrod; Aspirin; Blotting, Western; Cells, Cultured; Cycloheximide; Dactinomycin; Endothelium, Vascular; Epoprostenol; Fibrin; Humans; Kinetics; Oligopeptides; Prostaglandin-Endoperoxide Synthases; Umbilical Veins

Recent observations raise the possibility that by binding thrombin, fibrin(ogen) could modulate prothrombin activation, perhaps by modulating thrombin-mediated activation of factors VIII and V. Factors VIIIa and Va are the cofactors of intrinsic tenase and prothrombinase, respectively. This study compared the profile of prothrombin activation in pooled normal plasma, defibrinated pooled normal plasma, and pooled normal plasma containing fibrin I. Prothrombin activation was initiated with three stimuli, as follows: (1) addition of a suspension of thrombin, CaCl2, and coagulant phospholipids to plasma; (2) contact activation followed by recalcification of plasma; and (3) addition of a suspension of crude rabbit brain tissue factor and CaCl2 to plasma. Each plasma sample contained 2 mmol/L gly-pro-arg-pro, which prevents the polymerization of fibrin. Prothrombin activation, indexed as the concentration of prothrombin activation fragment 1 + 2 produced, was quantitated by enzyme-linked immunosorbent assay. Fibrinogen significantly delayed the onset of prothrombin activation initiated by thrombin or by contact activation but not that initiated with tissue factor. In contrast, fibrin I in solution accelerated prothrombin activation initiated with thrombin or by contact activation. Although fibrinogen had no effect on intrinsic activation of factor IX or factor IX activation initiated by adding thrombin to plasma, it inhibited factor X activation initiated with either stimuli. We therefore conclude that fibrinogen can inhibit the activation of factor X and prothrombin. Second, fibrin I accelerates prothrombin activation. Fibrinogen and fibrin, therefore, have the potential to bioregulate blood coagulation.

Topics: Factor IX; Factor X; Fibrin; Fibrinogen; Humans; Oligopeptides; Prothrombin; Thrombin

The role of the local synthesis of thrombin in platelet recruitment and thrombus stabilization in heparinized blood was examined in vitro. Mural thrombosis was visualized and measured in a thin, rectangular, collagen-coated capillary under controlled rheological conditions by using fluorescence digital videomicroscopy and fluorescence microphotometry. Thrombin activity was inhibited in heparinized blood by the synthetic competitive inhibitor, D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (FPRCH2Cl), resulting in a marked reduction in the rate of platelet accumulation on collagen surfaces, indicating a role for thrombin in platelet recruitment. Similar although lesser effects were observed by reducing thrombin synthesis with antibodies to factors II and X. To decouple the role of thrombin in platelet recruitment by direct stimulation of platelet activity from its role in thrombus stabilization via fibrin formation, thrombosis was measured in heparinized blood treated with the tetrapeptide glycyl-prolyl-arginyl-proline, which inhibits fibrin monomer assembly into fibrin. The ultimate level but not the initial rate of platelet accumulation was reduced markedly, indicating a role for fibrin in thrombus stabilization against hemodynamic forces. Scanning electron micrographs demonstrated fibrin stands in the heparinized control samples but not in the heparinized samples with glycyl-prolyl-arginyl-proline. These results demonstrate a role for the local action of thrombin synthesized on the surfaces of thrombi even under conditions when the thrombin exerts no bulk effect, such as under heparin anticoagulation. Furthermore, this role appears to be a result of both platelet recruitment and thrombus stabilization.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Antithrombins; Blood Platelets; Collagen; Factor X; Fibrin; Humans; Microscopy, Electron, Scanning; Models, Biological; Oligopeptides; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Prothrombin; Thrombin; Thrombosis

Topics: Blood Coagulation; Fibrin; Fibrinogen; Humans; Macromolecular Substances; Oligopeptides

Interactions between platelets and fibrin are important in hemostasis but often confused with platelet-fibrinogen interactions. Fibrin solubilized in NaBr-acetic acid formed polymers in neutral pH buffers. A stirred mixture of solubilized fibrin and washed human platelets at neutral pH range showed drastic reduction in turbidity and concomitant platelet adhesion onto newly formed fibrin strands. Optimally, more than 99% of platelets could be associated with fibrin, whereas only 3% of erythrocytes were trapped in the fibrin network under similar conditions. This platelet-fibrin interaction was fibrin concentration dependent and polymerization stage dependent. It preceded any detectable platelet release of serotonin. Gly-Pro-Arg-Pro (250 times in excess) retarded the kinetics of fibrin polymerization as well as that of platelet-fibrin interaction. However, factors affecting platelet activation showed little effect on platelet-fibrin interaction. Our results support the existence of a platelet-fibrin interaction that does not require platelet activation.

Topics: Adenosine Diphosphate; Alprostadil; Aspirin; Blood Platelets; Egtazic Acid; Fibrin; HEPES; Humans; Hydrogen-Ion Concentration; Kinetics; Nephelometry and Turbidimetry; Oligopeptides; Platelet Aggregation; Polymers; Serotonin

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.

Topics: Animals; Cattle; Factor XIII; Fibrin; Fibrinogen; Glutamine; Kinetics; Macromolecular Substances; Methylamines; Oligopeptides; Polymers; Putrescine; Transglutaminases

Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Fibrin; Fibrinogen; Humans; Kinetics; Oligopeptides; Protein Binding; Serum Albumin; Structure-Activity Relationship; Thrombin Time

A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl-L-prolyl-L-arginyl-L-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% (v/v) in 0.1 M Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 mM calcium chloride, and 0.25 mM glycyl-L-prolyl-L-arginyl-L-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 microliter of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl-L-prolyl-L-arginyl-L-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl-L-prolyl-L-arginyl-L-proline was directly measured using the assay.

Topics: Blood Coagulation Tests; Catalysis; Factor XIII; Fibrin; Fibrinogen; Hot Temperature; Humans; In Vitro Techniques; Oligopeptides; Thrombin; Transglutaminases

Thrombin cleavage of blood coagulation Factor XIII (a2b2) and fibrinogen was studied during in vitro clotting to determine the physiologic sequence of these events. First, the time course of fibrin formation and cleavage of Factor XIII was measured in platelet-rich plasma. Cleavage of fibrinogen was measured by using a radioimmunoassay for fibrinopeptide A. Conversion of trace amounts of radioiodinated a-chains of 125I-Factor XIII to thrombin-modified a-chains was measured in unreduced 10% sodium dodecyl sulfate-polyacrylamide gels. During spontaneous clotting, a similar percentage of 125I-Factor XIII and fibrinogen was cleaved at each time point. Visible gelation of polymerized fibrin monomer occurred when 24 +/- 8% of fibrinogen was cleaved and 21 +/- 6% of Factor XIII was converted to Factor XIII'. Thrombin cleavage of Factor XIII and fibrinogen was also studied in platelet-poor plasma to which thrombin was added. In order to measure Factor XIIIa activity, fibrin polymerization was completely inhibited by the addition of Gly-Pro-Arg-Pro. Factor XIIIa formation was measured by the incorporation of [3H]putrescine into casein. The concentration of added thrombin required to cleave 50% of fibrinogen and Factor XIII was 0.65 U/ml and 0.35 U/ml, respectively. The rate of cleavage of fibrinogen by thrombin was 43-fold greater than cleavage of Factor XIII. Lower Gly-Pro-Arg-Pro concentrations were used to determine the effects of incompletely inhibiting fibrin polymerization on cleavage of Factor XIII and fibrinogen. Thrombin cleavage of Factor XIII but not fibrinogen was dependent on the extent of fibrin polymerization. The more marked the degree of inhibition of fibrin polymerization, the slower the rate of Factor XIIIa formation. Thus, in platelet-rich plasma, thrombin cleavage of Factor XIII and fibrinogen are closely related events during spontaneous clotting. Furthermore, cleavage of Factor XIII during clotting is enhanced by fibrin polymerization in platelet-poor plasma.

Topics: Binding Sites; Blood Coagulation; Blood Platelets; Electrophoresis, Polyacrylamide Gel; Factor XIII; Fibrin; Fibrinogen; Humans; In Vitro Techniques; Oligopeptides; Thrombin; Time Factors; Transglutaminases

The effect of fibrin polymers on thrombin-catalyzed factor XIIIa formation was studied in afibrinogenemic plasma. Fibrin polymers derived from des A fibrinogen and des A,B fibrinogen increased sixfold the rate of thrombin-catalyzed factor XIIIa formation in the presence of EDTA. Calcium chloride accelerated factor XIIIa formation 14-fold in the presence of des A,B fibrinogen without increasing the rate of thrombin formation. Fibrinopeptides A and B had no effect on factor XIIIa formation in afibrinogenemic plasma. Des A,B fibrinogen reduced by 20- to 40-fold the thrombin concentration required to activate factor XIII. Glycyl-L-prolyl-L-arginyl-L-proline (gly-pro-arg-pro), a fibrin polymerization inhibitor, inhibited des A and des A,B fibrinogen from enhancing thrombin-catalyzed factor XIIIa formation. Gly-pro-arg-pro did not modify factor XIIIa formation in afibrinogenemic plasma and did not inhibit thrombin cleavage of the chromogenic substrate S-2238. These results demonstrate that fibrin polymers accelerate thrombin-catalyzed plasma factor XIIIa formation.

Topics: Afibrinogenemia; Calcium; Edetic Acid; Factor XIII; Fibrin; Fibrinogen; Humans; Kinetics; Oligopeptides; Thrombin; Transglutaminases

Interactions between platelets and fibrin have been visualized by phase contrast, epifluorescence, and scanning electron microscope examination of clots formed with dansylcadaverine-labeled fibrin and gel-filtered platelets. After thrombin activation, the platelets appeared as fluorescent aggregates with bridging strands of fibrin; formaldehyde-fixed platelets were not fluorescent under the same experimental conditions. Scanning electron micrographs demonstrated that thrombin-activated cells had numerous pseudopods to which the fibrin strands adhered; fixed platelets exhibited a smooth discoid appearance and did not interact with the clot. Platelets trapped in clots formed with Batroxobin (Pentapharm) (platelets are not activated by Batroxobin as confirmed by light-scattering aggregometry measurements) remained as nonfluorescent, discoid cells, whereas platelets first activated by adenosine diphosphate formed brightly fluorescent aggregates. Light-scattering data of thrombin activation (0.2 U/mL) indicated that preincubation of platelets with 0.1 mmol/L prostaglandin E1 (PGE1) prior to addition of thrombin decreased the extent and rate of platelet shape change and resulted in 100-fold slower aggregation. Clots formed in the presence of PGE1 revealed decreased fluorescence intensity and fewer platelet-fibrin contacts. Gly-Pro-Arg-Pro, which blocks fibrinogen binding and fibrin assembly, was also effective in blocking platelet-fibrin interactions. These results indicate that platelet activation is a prerequisite for attachment of platelets to fibrin.

Topics: Adenosine Diphosphate; Adult; Alprostadil; Batroxobin; Blood Platelets; Epoprostenol; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Light; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Oligopeptides; Platelet Aggregation; Prostaglandins E; Scattering, Radiation; Thrombin

Because fibrin, formed at a site of vessel wall injury, is involved in the formation and stabilization of a platelet aggregate or thrombus, we have studied reactions of fibrin with rabbit platelets. Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was used to prepare soluble fibrin. Fibrin alone did not cause aggregation of washed platelets, but addition of ADP caused aggregation and deaggregation identical to those observed in the presence of fibrinogen. Specific binding of 125I-fibrin to ADP-stimulated platelets was similar to that of 125I-fibrinogen, but 125I-fibrin did not dissociate, even in the presence of high concentrations of apyrase. High non-specific binding of 125I-fibrin was observed that was not associated with aggregation. EDTA, prostaglandin E1 (PGE1) and creatine phosphate/creatine phosphokinase prevented ADP-induced aggregation in the presence of fibrin and caused rapid deaggregation when added after ADP. They also inhibited 125I-fibrin binding when added before ADP, and EDTA or PGE1 caused partial dissociation of bound 125I-fibrin. In vivo, fibrin may bind to stimulated platelets, polymerize, form a gel, and interact with components of the plasma, the platelet aggregate, and the exposed subendothelium.

Topics: Adenosine Diphosphate; Alprostadil; Animals; Apyrase; Blood Platelets; Creatine Kinase; Edetic Acid; Fibrin; Fibrinogen; Oligopeptides; Phosphocreatine; Platelet Aggregation; Prostaglandins E; Rabbits

Steady state kinetic parameters were evaluated for the hydrolytic release of fibrinopeptides A and B (FPA and FPB) from human fibrinogen by human thrombin at pH 7.4, 37 degrees C, and gamma/2 0.15. At low concentrations of fibrinogen (less than 0.4 microM), the release of FPA from A alpha-chains was first order with respect to both the concentration of fibrinogen A alpha-chains and thrombin. The second order rate constant yielded a value of 11.6 (+/- 0.3) X 10(6) M-1 S-1 for the specificity constant (kcat/Km) for this process. Values of 84 (+/- 4) S-1 and 7.2 (+/- 0.9) microM were evaluated for kcat and Km for the thrombin-catalyzed release of FPA from normal human fibrinogen. The amino acid replacement ArgA alpha 16 leads to His present in fibrinogen Petoskey was shown to result in a 160-fold decrease in the specificity constant for hydrolysis at A alpha 16 and concomitant release of FPA. A kinetic analysis for determination of the sequentiality of release of fibrinopeptides was presented. It indicated that at least 97% of FPB was released after FPA. The specificity constant for release of FPB from intact fibrinogen (if it occurs) was less than 3% of that for release of FPA and less than 10% of that for release of FPB from des-A fibrinogen. The specificity constant for the release of FPB from des-A fibrinogen was 4.2 (+/- 0.2) X 10(6) M-1 S-1. The polymerization inhibitors EDTA and Gly-Pro-Arg-Pro inhibited release of FPB but not FPA. These observations are consistent with the generally accepted view that the predominant pathway for the conversion of normal human fibrinogen to fibrin is one wherein FPA is released, des-A fibrinogen polymerizes, and then FPB is released.

Topics: Edetic Acid; Fibrin; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; Kinetics; Mathematics; Oligopeptides; Thrombin

Topics: Adenosine Diphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Cytoplasmic Granules; Fibrin; Fibrinogen; Oligopeptides; Platelet Aggregation; Platelet Membrane Glycoproteins; Rabbits; Receptors, Cell Surface; Thrombin

The method for preparing thrombin-degranulated platelets has been modified to avoid the use of plasmin or successive treatments with small amounts of thrombin, while still achieving more than 90% release of platelet amine storage granule contents. It was necessary to prevent the fibrinogen released from the platelets during thrombin treatment from forming an insoluble fibrin mesh that could trap the platelets and hinder their deaggregation. To accomplish this we have treated rabbit platelets with 0.73 U/ml of thrombin for 1 min in the presence of the synthetic peptide, Gly-Pro-Arg-Pro, which prevents the polymerization of fibrin molecules. We have demonstrated that it also prevents 125I, initially added as 125I-fibrinogen, from associating with the platelets in a form that was not removed by centrifuging and washing during the preparation of thrombin-degranulated platelets, and we infer that products formed from the fibrinogen released from the platelets would also be prevented from associating with them. Thrombin-degranulated platelets prepared by this method have lost 92% of their granule contents and they can be washed and resuspended. These platelets aggregate normally upon stimulation with thrombin, adenosine diphosphate (ADP), or arachidonate. Thus, Gly-Pro-Arg-Pro is useful in preparing thrombin-degranulated platelets for studying platelet reactions without the complicating effects of released materials such a ADP and fibrinogen.

Topics: Animals; Blood Platelets; Cytoplasmic Granules; Dose-Response Relationship, Drug; Fibrin; Fibrinogen; Oligopeptides; Platelet Aggregation; Rabbits; Serotonin; Thrombin

Topics: Fibrin; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Humans; Oligopeptides; Peptides; Spectrophotometry; Thrombin