Dysfibrinogenemia and multiple sclerosis: spuriously associated or causally linked?

BACKGROUND: The inherited dysfibrinogenemias comprise rare congenital coagulation disorders which are clinically characterized by bleeding diathesis and, in occasional patients, by thrombotic tendency or combined bleeding-thrombotic events. In recent years, accumulating evidence suggested that fibrinogen has a critical role in the pathogenesis of neuroinflammatory disorders, including multiple sclerosis. We describe the presentation and long-term follow-up of a patient with inherited dysfibrinogenemia and concomitant clinical and laboratory evidence of demyelinating disease.   Case description:  A 16-year-old male patient presented in 2003 with bilateral sensory symptomatology preceded by an episode of epistaxis. His past medical history included episodes of spontaneous nosebleeds as well as Duane syndrome and mild atrophy of the right upper limb. Coagulation testing of the patient and his asymptomatic father revealed in both the presence of a clotting defect, consistent with inherited dysfibrinogenemia (named Fibrinogen Thessaloniki). Within seven months, the patient presented with a new episode of motor semiology whereas serial brain magnetic resonance imaging (MRI) scans revealed T2 lesions with bilateral distribution, some of which with gadolinium enhancement. The cerebrospinal fluid examination disclosed the presence of oligoclonal bands in the central nervous system compartment. The patient was started on azathioprine (2.5 mg/kg/24h) which led to clinical and radiological stabilization for nine years. In 2013, the dose of azathioprine was reduced, due to an elevation of his amylase levels, resulting in radiological deterioration with an increased T2 lesion load. The reinstitution of azathioprine at therapeutic doses led to radiological improvement and clinical stability as of today. CONCLUSION: The described case of inherited dysfibrinogenemia and concomitant multiple sclerosis provides speculative evidence for a causal link, rather than a chance association, between these two entities. Further studies are warranted to corroborate this hypothesis in experimental and clinical settings. HIPPOKRATIA 2017, 21(1): 49-51.

Evaluation of fibrinogen self-assembly: role of its αC region.

BACKGROUND: Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood. OBJECTIVE: To investigate hydrophobic surface-induced fibrinogen aggregation. METHODS: Contact and lateral force scanning probe microscopy, yielding topography, image dimensions and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non-enzymatic conditions by adsorption on a trioctyl-surface monolayer (trioctylmethylamine) grafted onto silica clay plates. RESULTS: A more than one molecule thick coating was generated by adsorption on the plate from 100 to 200 µg mL⁻¹ fibrinogen solutions, and three-dimensional networks formed from 4 mg mL⁻¹ fibrinogen incubated with uncoated or fibrinogen-coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from the surface ranged from approximately 3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking αC-domains was used as a coating or was incubated with intact fibrinogen-coated plates, or when the latter plates were sequentially incubated with anti-Aα529-539 mAb and intact fibrinogen. When an anti-Aα241-476 mAb was used instead, fine, long fibers formed. Similarly, sequential incubations of fibrinogen-coated plates with recombinant αC-domain (Aα392-610 fragment) or αC-connector (Aα221-372 fragment) and fibrinogen resulted in distinctly fine fiber networks. CONCLUSIONS: Adsorption-induced fibrinogen self-assembly is initiated by a more than one molecule-thick surface layer and eventuates in three-dimensional networks whose formation requires fibrinogen with intact αC-domains.

Mutations on fibrinogen (gamma 316-322) are associated with reduction in platelet adhesion under flow conditions.

In this paper we report on studies of platelet adhesion to several fibrinogen gamma chain variants under physiological flow conditions. Reduced platelet adhesion was found to patient dysfibrinogen Vlissingen and its recombinant form (deletion of gamma 319-320). Furthermore, substitutions of the amino acids 318, 320, or both in the recombinant fibrinogen gamma chain showed a strong decrease in platelet adhesion under flow conditions in our perfusion system. Antibodies raised against peptides covering these sequences inhibited platelet adhesion completely, which suggested that the gamma 316-322 sequence could be involved in platelet adhesion in flowing blood.

Antifibrinogen IgG, fibrinogen, and Clq complexes circulating in a hypodysfibrinogenemic proband. Isolation, stoichiometry, and partial characterization.

Circulating antifibrinogen antibodies have been reported in rare afibrinogenemic propositi, apparently occurring following fibrinogen replacement therapy, but immune complexes have not been described. In this report we describe circulating immune complexes formed by a monoclonal antifibrinogen IgG in a heterozygous hypodysfibrinogenemic (A alpha 16 Arg-->Cys) proband. Estimated by partial protein sequence and by other analyses, each immune complex consisted of one fibrin(ogen), one C1q, and 3-4 IgG molecules. The complexes were cryoprecipitable, a property also displayed by mixtures of proband IgG and normal fibrinogen. Indicating that both D and E domains were necessary for this behavior, cryoprecipitability was abolished by preincubation of the isolated IgG with either isolated normal fibrinogen fragment D100 or E. Consistent with the crossreactivity of the IgG with normal and mutant fibrinogen, the results suggest that the primary epitope resides on a D-E locus on the fibrin polymer formed by normal and abnormal molecules containing the uncleaved (or mutant) peptide A.

A functional assay suggests that heterodimers exist in two C-terminal gamma-chain dysfibrinogens: Matsumoto I and Vlissingen/Frankfurt IV.

Because it contains three pairs of polypeptides, fibrinogen isolated from heterozygous individuals is expected to be a mixture of homodimers and heterodimers. Nevertheless, heterozygous individuals with only homodimers have been identified. We synthesized two recombinant fibrinogens with the mutations from fibrinogen Vlissingen/ Frankfurt IV (gamma(delta)319, 320) and Matsumoto I (gammaD364H), both identified in heterozygous individuals. We found that polymerization of these fibrinogens was undetectable in 30 min; polymerization of a 1:1 mixture of variant and normal fibrinogen was the same as polymerization of a 1:1 mixture of buffer and normal fibrinogen; polymerization of either plasma fibrinogen was markedly impaired when compared to the 1:1 mixture of the respective variant and normal fibrinogens. We conclude that each plasma fibrinogen is a mix of homodimers and heterodimers, such that the incorporation of heterodimers into the fibrin clot impairs polymerization. We suggest that incorporation of heterodimers can induce clinical symptoms.

The receptor for the globular "heads" of C1q, gC1q-R, binds to fibrinogen/fibrin and impairs its polymerization.

The 33-kDa cellular C1q binding protein, designated gC1q-R was previously shown to bind a number of plasma proteins involved in the coagulation and kinin systems. This study demonstrates the interaction between recombinant gC1q-R and fibrinogen. Using enzyme-linked immunosorbent assays, biotinylated gC1q-R was found to bind to microplate-immobilized fibrinogen in a manner which was specific and inhibited by excess soluble fibrinogen or polyclonal antibodies directed against either gC1q-R or fibrinogen. Moreover, gC1q-R inhibited fibrin polymerization in a dose-dependent manner. Reptilase induced fibrin clot formation was completely inhibited by gC1q-R at a 2:1 molar ratio (gC1q-R:fibrinogen), and repolymerization of thrombin induced fibrin monomers was similarly abrogated. At equivalent molar concentrations, gC1q-R appeared to be a more potent inhibitor of fibrin polymerization than fibrinogen, a well-known inhibitor. Moreover, in the presence of both gC1q-R and soluble fibrinogen, the effect of each inhibitor on fibrin polymerization was additive. When plasmin derived fibrinogen degradation products, including the C-terminal D domain (D-100) or the N-terminal E domain, were immobilized on microtiter plates, gC1q-R bound to fibrinogen fragment D-100, but not to fragment E. Further digestion of fibrinogen fragment D-100 by plasmin to fragment D-60 resulted in loss of gC1q-R binding. Thus, gC1q-R binds to the D domain of fibrinogen/fibrin, and the carboxyterminal segment of at least the fibrinogen/fibrin gamma chain appears important for this interaction. These observations may suggest a potential role for gC1q-R in modulating fibrin formation particularly at local sites of immune injury or inflammation.

Fib420, the novel fibrinogen subclass: newborn levels are higher than adult.

Fib420 is a recently identified subclass of normal human fibrinogen in which two extended alpha chain isoforms (alphaE) replace the common alpha chains, yielding a molecule (ca. 420 kD) which is larger than the more abundant 340-kD form. Evidence for preservation of this subclass throughout vertebrate evolution suggests it performs some as yet unidentified vital function. A survey was undertaken to establish the range of plasma Fib420 levels in normal, healthy adults and in placental cord (fetal) blood. For measuring Fib420, a quantitative Western blot assay was developed using monoclonal antibody against the exon-VI encoded C-terminus of the molecule's unique alphaE chain. This alphaE chain signal was normalized to that of the beta chain, common to both fibrinogen forms. Analysis of plasma samples from the adult and newborn cohorts (n = 25 each; total fibrinogen ca. 2.6 mg/mL in both) revealed a statistically significant difference, with a mean level of 100 +/- 28 microg/mL in the neonate compared to 34 +/- 7 microg/mL in the adult. On average, 1 out of every 100 fibrinogen molecules in adult plasma belongs to the Fib420 subclass. Unlike in the newborn, adult Fib420 levels remained the same over a wide range of total plasma fibrinogen. The striking difference observed between these two cohorts suggests a changing developmental expression of the Fib420 subclass and a homeostatic control operating in later stages of life.

Platelet adhesion to late fibrinogen degradation products.

Evidence is emerging for the regulation of platelet function at sites of vascular injury or thrombosis by multiple platelet recognition sites in fibrinogen. This study examined the interaction of platelets with immobilized fibrinogen degradation products, fragments D and E. A 60 kDa D fragment (D60) and 30 kDa fragment E supported the adhesion of activated platelets in a static system, despite the absence of gamma chain 400-411 dodecapeptide and RGD sequences. Moreover, platelet adhesion to these fragments was incompletely inhibited by EDTA. In the absence of divalent cations, ADP-stimulated platelet adhesion to fragments D60 or E constituted 31 +/- 12% and 33 +/- 10% (mean +/- SD,n = 23) of adhesion to intact fibrinogen in the presence of divalent cations, respectively. This EDTA-resistant adhesion was distinctly modulated by thrombin which preferentially supported platelet adhesion to fragment E, and chymotrypsin which selectively supported platelet adhesion to fragment D60. Furthermore, two potent inhibitors of fibrinogen binding, the 10E5 monoclonal antibody directed against the GPIIb-IIIa complex and the RGDF peptide, inhibited EDTA-resistant platelet adhesion to fragment D60 but not to fragment E. These data suggest the presence of novel, non-RGD, non-dodecapeptide containing platelet recognition sequences in both fibrinogen D and E domains which support divalent cation dependent and independent platelet adhesion via potentially unique binding mechanisms.

The dimeric Aalpha chain composition of dysfibrinogenemic molecules with mutations at Aalpha 16.

In the last stage of fibrinogen synthesis, two Aalpha-Bbeta-gamma half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aalpha16 R-->H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA*, FPA*) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aalpha16 R-->H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R-->C (four) or R-->H (three) Aalpha16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot ('N-DSK') cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve 'N-DSK' components with two FPA*'s (N-DSK homodimer), one FPA* (des A N-DSK heterodimer), or no FPA's (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aalpha chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aalpha chain are produced, both Aalpha chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.

Plasma cryoprecipitation studies: major increase in fibrinogen yield by albumin enrichment of plasma.

The present studies compared fibrinogen yields of cryoprecipitate (Cr) obtained under differing conditions, and focused on yields from albumin enriched plasma. Addition of human albumin to fresh plasma collected into CPDA-1, citrate, or heparin (4 U/ml) resulted in an average of 2.8 fold (+/- 0.34 SD, n = 17) increase in yields of Cr fibrinogen. This albumin effect was shown with undefatted and defatted albumin, fibrinogen yields increasing in the range of 2-6 g of albumin added/dl of plasma and plateauing thereafter. Similarly increased were yields of fibronectin, plasminogen and factor XIII, but not of factor VIII or of von Willebrand factor. By electrophoretic analyses, Cr fibrinogen from albumin enriched and that from untreated plasma did not differ. Fibrin related measurements disclosed that the albumin enhancement of fibrinogen yield did not result form increased fibrin formation in Cr. This enhancement was shown in plasma that had been enriched with soluble fibrin to increase its yield and in that which had been subjected to hirudin, to high ionic strength, or to dilution to decrease its Cr fibrinogen yield. The results suggest a water exclusion effect, inducing cryoprecipitation of otherwise soluble fibrin/fibrinogen complexes.

Case report: treatment of an intracranial arteriovenous malformation in a patient with complicated hemophilia.

The authors describe a young man with hemophilia complicated by chronic hepatic dysfunction, hypodysfibrinogenemia, and immune thrombocytopenia that resulted in a complex coagulopathy. The patient had a ruptured occipital arteriovenous malformation. The malformation was managed by temporary correction of the coagulopathy using cryoprecipitate, platelet transfusions, and plasmapheresis with fresh frozen plasma replacement. The patient underwent staged preoperative embolization followed by surgical excision of the lesion. Hemostasis was acceptable during the neurointerventional and subsequent surgical management, and no complications of coagulopathy occurred. Plasmapheresis may provide effective preparation for patients with hemophilia and complex coagulation abnormalities who require neurosurgical intervention.

A unique property of a plasma proteoglycan, the C1q inhibitor. An anticoagulant state resulting from its binding to fibrinogen.

The C1q inhibitor, C1qI, an approximately 30-kD circulating chondroitin-4 sulfate proteoglycan, displayed concentration-dependent prolongation of plasma and fibrinogen solution clotting times. Under factor XIIIa catalyzed cross-linking conditions and maximum C1qI concentrations, minor amounts of clot formed displaying complete gamma-gamma dimer formation but virtually no alpha-polymer formation. The anticoagulant effect was undiminished by its binding to C1q, by increased ionic strength, and by CaCl2, but was abolished by incubation of C1qI with chondroitinase ABC. 125I-labeled C1qI bound to immobilized fibrinogen, fibrin monomer, fibrinogen plasmic fragments D1 and E, and fibrin polymers. Occupancy on the E domain required uncleaved fibrinopeptides together with another structure(s), and it did not decrease binding of thrombin to fibrinogen. Occupancy on the D domain did not decrease the fibrinogen binding to fibrin monomer. We conclude that the E domain occupancy impaired fibrinopeptide cleavage, and occupancy on the D domain impaired polymerization, both steric hindrance effects. C1qI binding to fibrinogen explains at least in part the well-known fibrin(ogen) presence in immune complex-related lesions, and the fibrinogen presence in vascular basement membranes and atheromata. We postulate that fibrin binding by resident basement membrane proteoglycans provides dense anchoring of thrombus, substantially enhancing its hemostatic function.

C1q inhibitor (chondroitin-4-sulfate proteoglycan): structure and function.

The serum C1q inhibitor (C1q INH) is a chondroitin 4-sulfate proteoglycan which is composed of several polyanionic components ranging in size from 21-750 kDa. Although the activity of C1q INH has been described in terms of its ability to precipitate C1q and inhibit its hemolytic activity, not much is known about either the mechanisms of its action or its role in health and disease. This report provides evidence that a 30 kDa core protein component of the proteoglycan macromolecule contains most of the C1q inhibitory activity. This inhibitory activity occurs as a result of C1q INH binding to the C1q "heads" (gC1q) as well as to the collagen "tail" (cC1q). What may be more significant in terms of perpetuation of inflammatory processes is the ability of C1q INH to moderately activate the classical pathway leading to C2 and C4 consumption. The binding of C1q INH to C1q is enhanced at low ionic strength, but significant binding does occur under physiologic conditions which makes it likely for the inhibitor to participate in inflammatory processes especially in microenvironments of high inhibitor concentration. Such elevated concentration does occur in patients with active rheumatoid arthritis and systemic lupus erythematosus either as a result of unregulated proteoglycan synthesis or disturbances in connective tissue metabolism. Another important function of serum C1q INH is its ability to prolong the clotting time of plasma and fibrinogen solutions containing or lacking CaCl2. This potent anticoagulant activity is again displayed by the 30 kDa putative protein core which specifically binds to both the E and D domains of fibrinogen. However, the epitope(s) on the 30 kDa which binds to C1q appears to be distinct from that which binds to fibrinogen. The known presence of proteoglycans on the basement membranes and other sites may explain at least in part the presence of fibrinogen in atheromatous lesions. Furthermore, by binding to fibrinogen, soluble C1q INH-and C1q-C1q INH complexes may limit fibrin gelation in inflammatory and tissue repair microenvironments.

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.

Fibrinogen anomalies and disease. A clinical update.

The precipitous increase in the number of structurally defined fibrinogen defects in recent years has resulted from application of high performance liquid chromatography in combination with peptide mapping and sequencing procedures. More recently, application of DNA sequence of polymerase chain reaction products has accelerated the pace of identification of mutations. Highly frequent defects are Arg substitutions, accounting for eight mutation sites substituted by Cys or His and less frequently by Ser. Amino acid substitutions at different positions on all three chains have pointed to possible structures with polymerization-related functions. Also, substitutions yielding consensus sequences resulted in extra glycosylations of the appropriate Asn in four different mutation sites; the impaired polymerization was reported associated with undue bleeding in two of these. Among informative defects have been those of homozygous probands with A alpha 16Arg----His and A alpha 16Arg----Cys in that failure of release of peptide A (but not of B), as shown with A alpha 16Arg----Cys, resulted in markedly delayed polymerization of such fibrin monomers, in general agreement with conclusions reached in studies of normal fibrin. This dysfunction, as well as the slow rate of release of A shown with A alpha 16Arg----His, was associated with clinically significant hemorrhagic diathesis (in the homozygous probands), consistent with the known physiologic importance of peptide A cleavage in normal hemostasis. Also, defects on the A alpha 17-19 sequence resulting in impaired polymerization are consistent with the known role of this segment in polymerization. Of similar interest have been defects within a B beta chain span encoded its exon 2. Two defects resulting in impaired polymerization and thrombin binding were associated with clinical thrombosis commencing in early life, and this lends strong support to other evidence suggesting a role in polymerization and in noncatalytic thrombin binding by this B beta chain segment. Thrombosis associated with A alpha 554Arg----Cys in a heterozygous proband with impaired tPA interaction is unique and may shed light on this poorly understood but important interaction among fibrin, plasminogen, and tPA. A group of different defects within the gamma 275-375 sequence have pointed to a polymerization role, evidenced by delayed gelation and impaired binding of mutant D to normal fibrin E. An unusual example is a 15 residue insertion between gamma 350 and 351 resulting in impaired polymerization, gamma chain crosslinking, and platelet aggregation support and is associated with hemorrhagic diathesis and poor healing.(ABSTRACT TRUNCATED AT 400 WORDS)

Autologous whole plasma fibrin gel. Intraoperative procurement.

Fibrin glue is a relatively recent addition to the armamentarium of hemostatic agents for surgical use. Its efficacy has been repeatedly demonstrated in almost all surgical disciplines and subspecialties. Its use in the United States has been limited because of the risk of viral transmission associated with the use of human plasma. Previous authors have described techniques that limit this risk, but they are frequently impractical, expensive, or cumbersome. We describe the use of patients' own fresh plasma to make fibrin gel at the operative field. It provided hemostasis at least as good as that from heterologous plasma glue in 40 cardiac surgical patients. Autologous whole plasma fibrin gel is inexpensive and safe and eliminates the risk of viral transmission associated with glue derived from heterologous donor plasma.

Anticoagulant albumin fragments that bind to fibrinogen/fibrin: possible implications.

The present studies describe an inhibitory effect on fibrin polymerization by albumin fragments. When added to blood or plasma, SCMF or unreduced albumin CNBrF delayed clot formation, in sharp contrast to their acceleration of clotting of fibrinogen solutions. CNBrF inhibition was less marked than that of SCMF. The latter consistently prolonged the lag phase and decreased the opacity of fibrin in plasma, effects that could not be abolished by EDTA or by calcium chloride. Clots formed lacked elasticity in that clotting times were undetectable by mechanical probe in the absence of calcium. Estimated by clot free liquor, PRP clots decreased in size at much slower rates than controls and at complete retraction their volume remained at least threefold higher that of controls (n = 6). When fibrinogen was isolated from plasma or fibrinogen (approximately 5 mg/ml) solutions containing SCMF 1 to 5 mg/ml four SCMF coisolated with fibrinogen (n = 3 and n = 4, respectively), assessed by SDS-PAGE, and these could not be dissociated from fibrinogen by size exclusion chromatography (n = 2). Such fibrinogen isolates displayed prolonged clotting times, decreased clot opacity, and similarly abnormal reaggregation of their solubilized fibrin. In other experiments, limited human neutrophil elastase digestion produced large albumin fragments of which, examined unreduced, several fragments also bound to fibrin(ogen) and displayed this anticoagulant property (n = 2). These and related results suggest that the anticoagulant property is attributable at least in part to the largest SCMF.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinogen Stony Brook II: partial characterization of a heterozygously transmitted peptide A anomaly.

We describe preliminary studies of a new familial dysfibrinogen, fibrinogen Stony Brook II, present in a propositus and his mother. Both presented with a history of unexplained and chronic joint swelling following trauma, and the propositus suffered recurrent knee haemarthroses following arthroscopic surgery. Prolonged plasma thrombin times required a 3-5 fold excess of normal plasma for correction. Isolated fibrinogen displayed prolonged clotting times and delayed onset of clot turbidity. Also, an abnormal peptide could be released by thrombin but not by batroxobin along with approximately half the expected amounts of normal A peptide. Assessed by its thrombin release and by its early HPLC retention position, the abnormal peptide suggests a possible A alpha-16-Arg----His substitution. The data suggests an association in these probands between this heterozygously transmitted anomaly and the apparently impaired healing in hypovascular sites.

Fibrinogen Stony Brook, a heterozygous A alpha 16Arg----Cys dysfibrinogenemia. Evaluation of diminished platelet aggregation support and of enhanced inhibition of fibrin assembly.

Assessed by high performance liquid chromatographic and amino acid sequence determinations, approximately one half (n = 4) of A peptide in fibrinogen Stony Brook (phi SB) contained the A alpha 16Arg----Cys substitution. To examine its functional behavior, mutant molecule-rich soluble subfractions that partly or fully lacked their normal A peptide were obtained from cryoprecipitates or from incoagulable material, respectively. Such subfractions consistently induced a more pronounced decrease (n = 3) in the turbidity of normal polymerizing fibrin than that induced by normal fibrinogen, by whole phi SB (n = 4) or by fibrinogen from an unrelated homozygous proband. These subfractions also exhibited decreased (12-50% of normal controls, fibrinogen 30-590 nM, n = 5) ADP-induced aggregation support of gel-sieved platelets, a decrease not demonstrable by whole phi SB, by fibrinogen from the homozygous proband, or by enrichment of the latter with normal soluble fibrin. A single isolate displaying diminished platelet aggregation support was 125I-labeled and examined further. It exhibited decreased binding to platelets, and Scatchard analysis indicated decreased binding affinity but normal maximum binding. We infer that phi SB contained heterodimers that exhibited these distinct functional properties when their normal A peptide had been cleaved.