Fibrinogen Molecule Research Articles

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment gamma316-322, part of the fibrin-specific region.

The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12-carboxy-terminal residues of the gamma-chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the gamma-chain segments gamma318-320 and gamma408-411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to gammaDelta408-411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: gammaDelta319-320, gamma318Asp-->Ala and gamma320Asp-->Ala. Furthermore, antibodies raised against the sequences gamma308-322 and gamma316-333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment gamma316-322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin-specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

Fibrinogen Hillsborough: a novel γGly309Asp dysfibrinogen with impaired clotting

We present a novel gamma-chain dysfibrinogen that was discovered in a 32-year-old asymptomatic man admitted to the hospital after a car accident. He presented with a low fibrinogen concentration, 0.5 mg/mL, and a prolonged thrombin clotting time, 58 seconds. Analysis of purified fibrinogen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a gamma-chain variant with an apparently higher molecular weight. Isoelectric focusing (IEF) demonstrated an anodal shift in the banding pattern of the chains and electrospray ionization mass spectrometry (ESIMS) showed a 27-Da increase in the average mass of the unresolved variant and normal gamma chains. DNA sequence analysis showed a heterozygous mutation of GGC (Gly)-->GAC (Asp) at codon 309 of the gamma chain gene. This Gly--> Asp substitution was consistent with the charge change shown by IEF as well as the mass change identified by ESIMS. Functional analysis revealed that thrombin-catalyzed polymerization occurred with a longer lag time, lower rate of lateral aggregation, and similar final turbidity compared to normal and that factor XIII cross-linking was normal. The polymerization results suggest that residue gamma309 is necessary for proper alignment of fibrinogen molecules, specifically in protofibril formation and D:D interactions. gammaGly309 is highly conserved and x-ray structures support the conclusion that the lack of a side chain at this position helps facilitate the close contact between abutting gammaD domains of condensing fibrin monomers during polymerization.

Binding strength and activation state of single fibrinogen-integrin pairs on living cells.

Integrin activation states determine the ability of these receptors to mediate cell-matrix and cell-cell interactions. The prototypic example of this phenomenon is the platelet integrin, alphaIIbbeta3. In unstimulated platelets, alphaIIbbeta3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables alphaIIbbeta3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified alphaIIbbeta3 or alphaIIbbeta3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80-100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single alphaIIbbeta3 molecules, indicating that there are only two states of alphaIIbbeta3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation/inactivation state.

Allosteric Effects Potentiating the Release of the Second Fibrinopeptide A from Fibrinogen by Thrombin

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen. When choline was substituted for sodium to transform thrombin to its slow form, the maximal levels of alpha-profibrin rose to those expected for independent release of the two FPA. Thus, it is only the fast thrombin that releases the second FPA fast, and that fast release only occurs when both FPA are present because of a partial coupling of its release with that of the first FPA. The release of the FPA from purified alpha-profibrin with the first FPA already missing is no faster than the release of any FPA. Surprisingly, we also found that slow thrombin became increasingly transformed to a fast form in the absence of sodium when the fibrinogen was elevated to high concentrations. This potentiation by concentrated fibrinogen also occurs with the recombinant mutant thrombin (Y225P), which is otherwise slow in both the presence and absence of Na(+). The potentiation of thrombin by fibrinogen must be short-lived so that the thrombin reverts to its slow acting form in the interim among encounters with other fibrinogen molecules in dilute fibrinogen solutions lacking Na(+), whereas at high fibrinogen concentrations the thrombin encounters other molecules before it reverts back to the slow form.

Laboratory diagnosis of dysfibrinogenemia.

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in abnormal fibrinogen function. It can be inherited or acquired. The inherited form is associated with increased risk of bleeding, thrombosis, or both in the same patient or family. Traditionally, dysfibrinogenemia is diagnosed by abnormal tests of fibrin clot formation; the thrombin time and reptilase time are the screening tests, and the fibrinogen clotting activity-antigen ratio is the confirmatory test. The inherited form is diagnosed by demonstrating similar laboratory test abnormalities in family members, and if necessary by analysis of the fibrinogen protein or fibrinogen genes in the patient. The acquired form is diagnosed by demonstrating abnormal liver function tests and by ruling out dysfibrinogenemia in family members. This article reviews the laboratory testing of dysfibrinogenemia and presents an algorithm for sequential test selection that can be used for diagnosis.

Thrombus Formation with Rehydrated, Lyophilized Platelets

Stored human platelets are frequently used in hemorrhagic emergencies, but have limited immediate utility for controlling bleeding due to storage lesion and are frequently contaminated with microorganisms. The development of paraformaldehyde-treated, lyophilized and rehydrated (RL) platelets, which are sterile and have a prolonged shelf life (years), ameliorate the efficacy and sterility problems with stored platelets. RL platelets have been shown to have many native functions of fresh platelets in vitro and to mediate hemostasis in vivo in large animal models of hemorrhagic shock and cardiopulmonary bypass induced platelet dysfunction. To further evaluate the functional properties of this transfusion product, we studied the role of RL platelets in three aspects of thrombus formation and lysis. First, the interaction between RL platelets and fibrinogen was investigated. The surface density of unligated GPIIb-IIIa on RL and fresh platelets were, respectively 30,000 and 70,000 molecules per cell as detected with the monoclonal antibody 10E-5. Freezing, lyophilization and rehydration steps in the preparation of RL platelets resulted in the surface presentation of 120,000 molecules of fibrinogen per cell from alpha granule sources. After ADP activation, RL platelets bound exogenous 125I-labeled fibrinogen in a dose-dependent manner with an affinity that is similar to that of fresh platelets and was inhibited by RGD peptides. 125I-Labeled fibrinogen binding to RL and fresh platelets, respectively, saturated at 14,000 and 32,000 molecules per cell. Scanning electron microscopic ultrastructural analysis showed that fibrin strands interacted with the surface of RL platelets in a normal manner. The second set of studies investigated the ability of RL platelets to catalyze and amplify the clot formation process in an activation-dependent manner. We showed that RL platelets undergo degranulation in fibrin in clots and functioned as thrombogenic surfaces for the generation of activated coagulation factors and fibrin generation. A final set of studies was performed to investigate fibrin of clots that contained RL platelets. RL platelet clots were lysed in the presence of tissue plasminogen activator with a similar time course as clots without platelets, and lysis occurred faster than when fresh platelets were included in the fibrin mass. The results of these three studies demonstrate that RL platelets are capable of mediating thrombus formation and do not inhibit lysis. Our results help explain how RL platelets restore hemostasis in vivo, and indicate that these cells might be a viable alternative to fresh stored platelets in transfusion medicine.

Influence of end products of plasmin-mediated hydrolysis of fibrinogen and fibrin (EF and Ef fragments) on fibrinogen cross-linking

We studied the influence of the end products of plasmin-mediated hydrolysis of fibrinogen and nonstabilized fibrin (EF and Ef fragments) on covalent cross-linking of fibrinogen molecules catalyzed by a fibrin-stabilizing factor (factor XIIIa). The data on elastic and dynamic light scattering reveal no difference in the spatial structure of covalently linked fibrinogen molecules in the presence of the hydrolysis end products EF and Ef. In contrast to the inactive fragment EF, fragment Ef significantly accelerates the enzymatic reaction. This is also confirmed by electrophoresis of the reduced samples indicating a relatively fast accumulation of gamma-dimers and A alpha-polymers as compared to the control samples. Possible molecular mechanisms of this effect are discussed.

Glanzmann's thrombasthenia: updated

Glanzmann's thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively more common platelet function defect in communities where consanguineous marriages are more frequent. On clinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gum bleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma or hemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count and morphology, a greatly prolonged bleeding time, absence of platelet aggregation in response to ADP, collagen, epinephrine, thrombin and to all aggregating agents which ultimately depend on fibrinogen binding to platelets for this effect, flow cytometry, studies of GPIIb-IIIa receptors on the platelet membrane surface using monoclonal antibodies. The present review describes some of the uncommon features of the disorders and the currently available options which the treating physicians should be aware of during the management of these patients. Although by definition all patients with Glanzmann's thrombasthenia have a virtually complete failure of platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins are present in normal or near normal amounts but are functionally defective. Understanding the pathophysiology of the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptors resulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GT management. It has now been established that different genetic mutations of either GPIIb or IIIa genes results in such a heterogeneity of thrombasthenia phenotype. Glanzmann's thrombasthenia is a paradigm for treating coronary artery disease patients with GPIIb-IIIa antibody and inhibitors. By using these medicines we create a temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicing cardiologist. As mutations for different variant forms of GT become known, our understanding of how GPIIb-IIIa molecules can be activated to act as a receptor for fibrinogen molecules will be increased. Such understanding undoubtedly will help us to devise better drugs with GPIIb-IIIa inhibitors. Molecular biology techniques have enabled us to equivocally detect heterozygote carriers who are clinically asymptomatic. However, there may be several laboratories in the developing world, which have no access to molecular biology techniques. Development of more robust techniques of quantitation of platelet receptors has enabled an accurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of the GPIIb-IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the less direct method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as 16 weeks of intrauterine life can be used for prenatal diagnosis of GT.

Mouse Models in Coagulation

In the past six years, analysis of numerous murine models, enabled by investigator-designed manipulation of the mouse genome, has gene-rated an explosion of new, and sometimes confounding, data. Mice with complete deficiencies in virtually all the known factors involved in hemostasis have been produced by targeted modification of the genes that encode the proteins that generate and regulate coagulation. Mice with altered factors or altered expression of normal factors have also been produced by transgenic techniques. In this review, we summarize the analyses of these mouse models and compare these results to those obtained from people with deficiencies in the same genes (Table 1). In-formation is available for all the models, but we will focus on those more completely characterized. We present the models in groups (Fig. 1), which places each factor into a single position within a simpli-fied model of hemostasis. The phenotypes of these genetically modified mice reflect a compilation of functions, some of which do not fit within this simplified model of hemostasis. For example, Factor X (FX) not only acts in concert with factor V (FV) to foster clot forma-tion but also acts in concert with tissue factor pathway inhibitor (TFPI) to limit clot formation. Additionally, we know that many factors have functions outside hemostasis; for example, a cleaved fragment of anti-thrombin III (ATIII) serves as an anti-angiogenic factor. Moreover, as shown remarkably by the analysis of these mouse models, many of these proteins also have a critical role in normal embryonic develop-ment (Fig. 2). Finally, genetic background significantly influenced the phenotype of some models, and we specify when this variable was examined. To provide a structure to consider analysis of these mice, we divided the hemostasis proteins into three categories: those that promote the generation of thrombin, those that participate in clot formation, those that curtail the generation of thrombin. To place these proteins in con-text, we begin with a brief overview of coagulation. Coagulation is initiated when injury to the blood vessel exposes tissue factor (TF) to circulating factor VII (FVII), resulting in the formation of TF/VII complexes. This complex activates factor X (FX), either directly or indirectly though FIX. Activated FX (FXa) converts prothrombin to thrombin. Thrombin catalyzes multiple events in coagulation by acti-vating the platelet integrin IIb3, which mediates fibrinogen- and von Willebrand factor (vWF)-dependent platelet interactions, and by trans-forming soluble fibrinogen molecules into a fibrin fiber matrix. Thrombin further amplifies its own generation by activating factors (FXI, FX, FIX) and cofactors (FV and FVIII) that promote TF-inde-pendent production of the active protease FXa. Anticoagulation is achieved through the inhibition of the various steps of thrombin gene-ration. TFPI forms a complex with TF, FVIIa and FXa to terminate the initiating step. Protein Z forms a complex with FXa and serves as a cofactor in the inhibition of FXa activity. Thrombomodulin (TM), an endothelial cell surface-bound protein, and thrombin complex to activate protein C (PC). With its cofactor protein S (PS), activated protein C (APC) degrades FVa and FVIIIa and thus limits generation of thrombin. The serpin, ATIII, forms complexes with FXa and thrombin to directly inhibit these proteases.

Novel Fibrinogen Truncation with Deletion of Bβ Chain Residues 440-461 causes Hypofibrinogenaemia

A 24-year-old male with hepatitis C was initially diagnosed with hypofibrinogenaemia during investigations prior to a liver biopsy. He had a low functional and gravimetric fibrinogen concentration of 1.0 mg/mL and DNA sequencing of all exons, exon-intron boundaries and promoter regions of the fibrinogen Aalpha, Bbeta, and gamma genes revealed a single heterozygous g-->a mutation at nucleotide 8035 of the Bbeta gene. This creates a premature stop at the Trp 440 codon and results in a 22-residue truncation of the Bbeta chain. Analysis of purified plasma fibrinogen by SDS PAGE, reverse phase HPLC and ESI MS, however, failed to detect any of the truncated chains in the plasma fibrinogen. The non-expression of aberrant molecules was further confirmed by functional analysis, which revealed normal fibrin polymerisation. The principal structural feature of the independently folding betaD domain is its five-stranded anti-parallel beta sheet. The deletion here of residues 440 to 461 removes the second strand from this sheet structure and appears to impact on the viability of the nascent chain and its ability to be incorporated into mature fibrinogen molecules. The mutation does not however provoke the formation of hepatic inclusion bodies.

Fibrinogen Assembly and Crosslinking on a Fibrin Fragment E Template

There is an ongoing controversy concerning whether crosslinked gamma chains in fibrin are oriented "transversely" between fibril strands or "end-to-end" along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, cross-linked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked gamma chains in fibrin under physiological conditions were also aligned end-to-end. To assess its validity we studied the assembly and organization of fibrinogen molecules on a des AB-fibrin fragment E (E-des AB) or a des A-fibrin fragment E (E-des A) template. We evaluated the roles of E polymerization sites E(A) and E(B), and D association sites gammaXL, Da, Db, betaC and alphaC in this process. E(A):Da interactions caused fibrinogen: E "DED" complexes to form, and markedly enhanced the gamma chain crosslinking rates of fibrinogen or des alphaC-fibrinogen. Fibrinogen crosslinking without added fibrin E was slower, and that of des alphaC-fibrinogen was still slower. These events showed that although alphaC domains promote fibrinogen fibril assembly and crosslinking, they contribute little to increasing the E(A):Da-dependent crosslinking rate. Electron microscopic (STEM) images of E-des AB and fibrinogen plus factor XIIIa showed single-, double-, and multistranded fibrils with interstrand DED complexes aligned side-to-side. This alignment was due to betaC:betaC contacts resulting from D subdomain rearrangements initiated by the E(B):Db interactions, and also occurred in mixtures of des alphaC-fibrinogen with E-des AB. In contrast, a mixture of fibrinogen and E-des A plus XIIIa revealed double-stranded fibrils with interstrand DED complexes in a half-staggered arrangement, an alignment that we attribute to crosslinking of gammaXL sites bridging between fibrils strands. These and other features of E-des A-based fibrinogen fibrils, including interstrand gamma chain bridges and early and extensive lateral fibril strand associations concomitant with accelerated gamma chain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned gamma chains.

Peroxynitrite-mediated modification of fibrinogen affects platelet aggregation and adhesion

The reaction of peroxynitrite with fibrinogen resulted in both structural modifications and altered biological properties of this glycoprotein. SDS-PAGE analysis of peroxynitrite-treated fibrinogen, performed under non-reducing conditions, showed some aggregated material on the top of the gel (5-10% of total staining bands) and the presence of nitrotyrosine. The amount of nitrotyrosine, detected by immunoassay with anti-nitrotyrosine antibodies, was dependent on peroxynitrite concentration. In comparison with native molecule, peroxynitrite-treated fibrinogen subjected to SDS-PAGE under reducing conditions revealed not only three bands corresponding to A f , B g and n chains, but the existence of additional high molecular bands probably due to the formation of dityrosine crosslinking between fibrinogen subunits. The different susceptibility in tyrosine nitration of fibrinogen subunits was also observed. The A f chain was the most intensely nitrated, while B g and n chains were nitrated much less? Peroxynitrite-treated fibrinogen in comparison with native molecule had a distinct capability to mediate platelet adhesion and aggregation. Both unstimulated and ADP-activated platelets showed a reduced ability to adhere to peroxynitrite-modified fibrinogen. The percentage of ADP-induced platelet aggregation decreased as a function of peroxynitrite-mediated modification of fibrinogen molecule.

Stabilizing Effects of Various Polyelectrolyte Multilayer Films on the Structure of Adsorbed/Embedded Fibrinogen Molecules: An ATR−FTIR Study

The structural changes in fibrinogen as a consequence of its adsorption onto the surface of or its embedding into the interior of poly(allylamine hydrochloride) (PAH) or poly(styrenesulfonate) (PSS) multilayers are investigated by means of attenuated total reflection Fourier transform infrared (ATR−FTIR) spectroscopy. It is found that both adsorption and embedding preserve the secondary structure of the fibrinogen molecules. Furthermore, the interactions of the polyelectrolytes with the protein molecules prevent their aggregation, especially in the embedded state, at room temperature. Thus, it seems that the structure and the biological activity of proteins adsorbed on or embedded in polyelectrolyte multilayers could largely be preserved, which opens up great perspectives in the design of new bioactive surfaces. The nature and the extent of the polyelectrolyte−protein interactions are further studied via analysis of the thermotropic responses of the different architectures. It is found that both PAH- and PSS-terminated polyelectrolyte multilayers can elevate the onset temperature of the structural changes in adsorbed/embedded fibrinogen molecules by about 5 °C as compared with that for fibrinogen in solution. These polyelectrolytes also broaden the thermally induced structural transitions in the adsorbed/embedded fibrinogen molecules. The magnitude of these thermally induced structural changes is polyelectrolyte- and architecture-dependent. Whereas multilayer PAH−fibrinogen and multilayer PSS−fibrinogen constructions exhibit roughly the same large-scale thermally induced structural changes, in all architectures where fibrinogen is embedded the scale of these structural changes is restricted. The restriction becomes stronger as the presence of PSS at the polyelectrolyte−fibrinogen interfaces increases (PAH−fib−PAH < PAH−fib−PSS ≈ PSS−fib−PAH < PSS−fib−PSS). In the PSS−fib−PSS arrangement, the secondary structure of fibrinogen as determined from its infrared spectrum changes only slightly up to 90 °C. The underlying processes of the thermally induced structural changes is, in addition, different for fibrinogen molecules adsorbed onto or embedded into PAH-terminated polyelectrolyte multilayers. A tentative model based on “encapsulation” of the embedded protein by the polyelectrolytes is proposed to explain the observed features.

Vascular biology of thrombosis: the role of platelet-vessel wall adhesion.

Arterial thrombosis is initiated after atherosclerotic plaque rupture. The ruptured plaque triggers a cascade of platelet-mediated events that result in the formation of a platelet-rich thrombus. Thrombus development ultimately leads to vessel occlusion, the precipitating event in most myocardial infarctions and many strokes. Arterial thrombosis involves a process of platelet adhesion, activation, and aggregation. Platelets first form a monolayer by adhering to blood vessel subendothelial matrix proteins (collagen and von Willebrand factor) via interactions with platelet membrane glycoprotein (GP) receptors. This interaction produces an intracellular signal that activates platelets to increase expression of a specific GP receptor, GPIIb/IIIa, with a high affinity for fibrinogen and other adhesion molecules. Binding of fibrinogen to adjacent platelets results in irreversible platelet aggregation and the formation of the platelet aggregate. Understanding of this process has led to considerable research into potential antiplatelet compounds. A number of effective antiplatelet drugs are now available, which affect different stages along the platelet adhesion/activation/aggregation pathway. These include aspirin, dipyridamole, and the adenosine diphosphate antagonists. Much effort is now focused on development of thrombin receptor antagonists. However, this is proving to be difficult. Considerable interest has surrounded a new class of platelet drugs, the GPIIb/IIIa receptor antagonists. These drugs have proved effective in short-term infusion trials. However, long-term studies of oral GPIIb/IIIa receptor antagonists have shown increases in mortality. It is believed that these antagonists may act as partial agonists and may increase the risk for thrombosis.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

The dysfibrinogenaemias.

The dysfibrinogenaemias.

The impaired polymerization of fibrinogen Longmont (Bβ166Arg→Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers

AbstractThis study identified a new substitution in the Bβ chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site “a.” Thus, the sites “A” and “a” can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166→Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils.

Platelet-fibrinogen interactions.

Binding of fibrinogen to GPIIb-IIIa on agonist-stimulated platelets results in platelet aggregation, presumably by crosslinking adjacent activated platelets. Although unactivated platelets express numerous copies of GPIIb-IIIa on their surface, spontaneous, and potentially deleterious, platelet aggregation is prevented by tightly regulating the fibrinogen binding activity of GPIIb-IIIa. Preliminary evidence suggests that it is the submembranous actin or actin-associated proteins that constrains GPIIb-IIIa in a low affinity state and that relief of this constraint by initiating actin filament turnover enables GPIIb-IIIa to bind fibrinogen. Two regions of the fibrinogen alpha chain that contain an RGD motif, as well as the carboxyl-terminus of the fibrinogen gamma chain, represent potential binding sites for GPIIb-IIIa in the fibrinogen molecule. However, ultrastructural studies using purified fibrinogen and GPIIb-IIIa, and studies using recombinant fibrinogen in which the RGD and relevant gamma chain motifs were mutated indicate that sequences located at the carboxyl-terminal end of the gamma chain mediates fibrinogen binding to GPIIb-IIIa. There is evidence that fibrinogen itself binds to regions in the amino terminal portions of both GPIIb and GPIIIa and that the sites interacting with the fibrinogen gamma chain and with RGD-containing peptides are spatially distinct. Nonetheless, there appears to be allosteric linkage between these sites, accounting for the ability of RGD-containing peptides to inhibit platelet aggregation and arterial thrombosis.

Crystal structure studies on fibrinogen and fibrin.

X-ray crystallography studies on fragments D and double-D from human fibrinogen and fibrin have revealed the details of knob-hole interactions between fibrin units, as well as the nature of the association at their ends. More recently, a lower-resolution structure of native chicken fibrinogen has provided details about the structure of the central domain, and particularly the arrangement of disulfide bonds. Parts of the fibrinogen molecule are so flexible that they have not been visualized in electron density maps. The elusive regions include the alpha C domain, the amino-terminal segments of the alpha and beta chains, and the carboxyl-terminal segments of the gamma chains. Nonetheless, when all the structural data are considered together, it is possible to construct a realistic model not only of a fibrinogen molecule but also of a fibrin protofibril.

Fibrinogen Longmont

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.