Afibrinogenemias and Dysfibrinogenemias

Dennis K. Galanakis

Congenital fibrinogen disorders have provided novel insights into structure/function relationships and into the central role of fibrinogen in hemostasis. They are generally classified as quantitative (type I, a- or hypofibrinogenemia) and qualitative (type II, dysfibrinogenemia) groups. Afibrinogenemia was first reported in 1920,¹ hypofibrinogenemia in 1935,² and dysfibrinogenemia in 1958.³ The first fibrinogen mutation (Detroit) was described in 1969⁴ using protein sequencing, and use of DNA sequencing was first applied in 1989 to identify fibrinogens Hershey II and Leogan.⁵ Subsequent application of mass spectrometry remains the preferred method to confirm results from genetic techniques. Aside from a 1974 compilation⁶ and an updated listing in http://www.geht.org/databaseang/fibrinogen/, recent reviews provided detailed updates of this steadily expanding area of investigation.⁷^,⁸^,⁹^,¹⁰ To date, afibrinogenemia accounts for 23%, hypofibrinogenemia for 26%, and dysfibrinogenemia for 51% of all three. Estimated from the present selection of mutations, dysfibrinogenemia accounts for 51%, afibrinogenemia for 23%, and hypofibrinogenemia for 26%. This relative frequency and and the ˜0.6% prevalence of apparent congenital hypofibrinogenemia among in a 5,746 Japanese patient population¹¹ suggest an over a prevalence of ˜2% for all three disorders, and a possibly similar prevalence in other populations. Also, with the present selection of mutations, afibrinogenemia and dysfibrinogenemia are most frequently caused by FGA and FGG mutations, 38% and 35%, respectively as compared to 27% by FGB. Apart from its hemostatic functions, fibrinogen binds to vascular endothelial and other cells, interacts with many extracellular matrix components (see Chapter 16), and enables placentation from the middle of first trimester to full term.¹²^,¹³^,¹⁴ Moreover, fibrin provides nonsubstrate binding for thrombin, serves as platform for assembly of fibrinolytic proteins, and is the preferred substrate for the protease plasmin.

AFIBRINOGENEMIA

Causative Mutations

This is an autosomal recessive disorder that often results from consanguinity. Mutations that cause afibrinogenemia (Table 54.1) are typically homozygous causing deficiency at the DNA or at the RNA level by affecting mRNA splicing or stability, or by affecting protein synthesis, assembly, or secretion.¹⁰^,¹⁵ Since the first mutation was identified in 1999,¹⁶ more than 80 novel mutations have been described. Null mutations are most common and include frameshift or large deletions, and early truncating nonsense, or splice site mutations. Less frequently, missense or late-truncating nonsense mutations allow synthesis of the corresponding chain but impair intracellular fibrinogen assembly and/or secretion.

Clinical and Laboratory Manifestations—Hemorrhage

Hemorrhagic diathesis commences during the neonatal period and in some cases at a later age.¹⁷ Umbilical cord bleeding occurs in up to 85%, and other common sites are gastrointestinal tract, skin, and genitourinary tract. There is an increased incidence of splenic rupture,¹⁸ and intracranial bleeding is the major cause of death. Hemarthrosis and/or muscle hematomas are reported in about half. In menstruating women, menometrorrhagia is common and spontaneous first-trimester abortion is typical.¹⁹ Predictably, clotting time is infinite, bleeding time is prolonged, and fibrinogen-dependent platelet aggregation is impaired.²⁰ Also, erythrocyte sedimentation rate is decreased,²¹ and delayed hypersensitivity skin test reaction sites do not develop the normal induration.²²

Thrombosis

Paradoxically, venous or arterial thrombosis occurs in some probands.²³ Thrombosis risks such as antithrombin III, protein C deficiency, or others, present in few probands,²⁴ are absent in most.²⁵ Among six unrelated probands,²⁵ arterial thrombosis sites included iliac, hypogastric, and other peripheral arteries. A prevailing explanation is that in the absence of fibrin, unsequestered thrombin induces vaso-occlussive platelet aggregate formation. This is based on evidence that fibrin sequesters thrombin,²⁶^,²⁷ and that in a fibrinogen knockout mouse model²⁸ spontaneously formed platelet aggregates embolized six times more frequently than those of wild-type controls and were often vaso-occlusive. Also, plasma levels of prothrombin activation peptides and of thrombin-antithrombin complexes are elevated in such probands, and both normalize following fibrinogen replacement.²⁹^,³⁰ Among eight unrelated probands with venous thromboembolism (VTE), the affected veins included portal vein, mesenteric vein, cerebral sinus, and others.²⁵ An intriguing feature is the association with fibrinogen infusion in five probands with venous and two with arterial thrombosis, suggesting a causative role by infused fibrinogen. Among possible explanations is substantial enrichment of plasma cryoprecipitate, and possibly other fibrinogen preparations, with soluble fibrin/fibrin(ogen) complexes³¹^,³² known to shorten fibrinogen clotting times.³³ Also, active thrombin was demonstrated in such complexes from a dysfibrinogenemic proband with recurring thrombosis.³⁴ Such thrombin may be released by proteolytic degradation of the complexes at the wound or other sites. Suggesting another possible mechanism, fibrinogen excess decreases plasminogen binding to fibrin polymers.³⁵

HYPOFIBRINOGNEMIA

Mutations that cause afibrinogenemia are usually homozygous and in a few cases compound heterozygous. They cause decreased plasma concentrations of normal fibrinogen when they are heterozygous (Table 54.2), often with decreased assembly and/or secretion of the variant fibrinogen. Substitutions AαC36G or R³⁶ disrupt formation of Aα36C-Bβ65C and Aα36C-γ23C bonds,³⁷ while AαC45Y³⁶ and C45F³⁸ disrupt formation of the AαC45-γ23C bond.³⁹ As detailed elsewhere,³⁶ these confirmed experimental data showing that these bonds are required for hexamer assembly and/or secretion. Among mutations in the FGB gene two missense, R255H⁴⁰ and D346Y,⁴¹ and one nonsense mutation, W470X,⁴² result in changes in the β-domain of the distal D nodule of fibrinogen, suggesting impaired secretion. A second nonsense mutation, Y71X,⁷ likely results in impaired assembly since the encoded chain lacks the β-domain of the distal D nodule. A single splice-site mutation at the 5′ site of FGB intron 6 (IVS6 + 1G > A) results in a frameshift after P319, adding 18 novel residues prior to a stop codon.⁴³ Like other mutations in this region (Table 54.2), variant fibrinogen may be assembled but is not secreted. Also not secreted is fibrinogen with γC153R.⁴⁴ In two unrelated probands with hemorrhagic diathesis,⁴⁵^,⁴⁶ substitution A82G is accompanied by a BβP265L in one and by an FGG splice-site mutation (IVS2 + 1G > A) in the other. Considered together, these γ chain variants are likely synthesized but not incorporated into hexamers.

Four mutations result in hypofibrinogenemia, hepatocellular intracytoplasmic inclusions, and progressive liver disease in the same proband (Table 54.2). Three include missense mutations, γG284R (Brescia),⁴⁷ γR375W (Aguadilla),⁴⁸ and γT314P (Al DuPont),⁴⁹ all occurring within or near the “a” pocket. Defective polymerization of fibrinogen Brescia was attributed to its hypersialylation, a characteristic of fibrinogen in liver disease.⁵⁰ The fourth, a γ346-350 del (Angers)⁵¹ occurring within the five-stranded beta sheet of the γC, results from deletion of FGG nucleotides 7690A-7704G which deletes the splice site at position 7703. This generates a new (donor) splice site at position 7688, resulting in the in-frame deletion of five amino acids (GVYYQ) 346 to 350. Interestingly, other heterozygous mutations in these fibrinogen regions result in either assembly and secretion (dysfibrinogenemia) or selective secretion of only normal molecules (hypofibrinogenemia) but no intracellular accumulation.⁵²

FIGURE 54.1 Schematic representation of functional regions of fibrinogen and fibrin. The C-terminal sequences of the γ_A and γ′ chains are amplified for clarity, showing the high affinity thrombin binding, platelet interaction, and factor XIIIa catalyzed cross-linking (XL) sites. Elsewhere on the D region are the ‘a’ and ‘b’ pockets, and the D:D interface indicating the sites between D s of two molecules that associate during fibril elongation. The lower left panel indicates the slight weakening of αC tethering to the E region by FpA release, reflecting the contributing role to the tether by intact FpA.¹³¹ The lower right panel illustrates the untethering and dissociation of αC s following FpB release.¹²⁸^,¹³²

Clinical Manifestations

Hemorrhagic manifestations are common when plasma fibrinogen levels are in the range of 0.1 to 0.5 g/L¹ (normal 1.50 to 3.50 g/L¹) by clotting assays. Symptoms include bruising, epistaxis, menometrorrhagia, miscarriage, and hemorrhage after trauma, surgery, or during pregnancy and parturition. By contrast, probands with plasma fibrinogen at diagnosis as low as 0.60 to 0.70 g L^-1 are usually asymptomatic. The moderately prolonged thrombin (TT) and reptilase (RT) times are corrected by equal mixtures with normal plasma. Similar results may be obtained when a variant fibrinogen comprises a minor fraction of plasma fibrinogen (hypodysfibrinogenemia).

DYSFIBRINOGENEMIA

Dysfibrinogenemia is an autosomal dominant disorder that comprises mostly heterozygous mutations that result in both variant and normal fibrinogen in circulation. At least 500 families are known to date. Predictably,⁵³ plasma fibrinogen consists of normal and abnormal homodimers and of heterodimers, and its level by clotting assays is typically decreased, less than half that by antigen assay which is usually in the normal range. In a minor proportion of mutations (Table 54.2), fibrinogen is decreased by both assays and it is known as hypodysfibrinogenemia.

Defects that Impair Conversion of Fibrinogen to Fibrin

Following fibrinopeptide cleavage, fibrin monomers self-assemble to form protofibrils that polymerize by lateral contact to form fibers and networks (FIGURE 54.2). The specific interactions resulting in normal fiber formation have been elucidated in x-ray crystallographic structures of fibrinogen fragments D and factor XIIIa-cross-linked
fragment D-D in the presence of peptides GlyProArgPro-amine (GPRPam) and GlyHisArgPro-amine (GHRPam), which mimic the N-terminal α and β sites of fibrin, respectively.⁵⁴^,⁵⁵^,⁵⁶^,⁵⁷ Substitutions occurring within these crystal structures have enabled correlations of clinical and biochemical data with the three-dimensional fibrinogen structure. Mutations in the E region (Table 54.2) impair one or more of the following functions: thrombin binding to fibrinogen, fibrinopeptide cleavage, and fibrin polymerization.

Table 54.1 Mutations resulting in afibrinogenemia

*FGA*
cDNA	Mature Chain	Type	Reference
4.1 kb del		Large del of exon 1	209
c.3-4insC	F17Lfs	Fs, exon 1	210
c.54+1G>A		Spl. site mutation, intron 1	211
c.54+3A>G		Spl. site mutation, intron 1	210
11 kb del		Large del, exons 2-6, recurrent	210
c.94G>T	G13X	Nonsense mutation, exon 2, escapes nonsense-mediated decay (NMD)	212
c.117Tdel	V21Wfs	Fs mutation, exon2, escapes NMD	212
C196-197InsT	S47Ffs	Fs mutation, exon 3	211
c.209T>G	M51R	Missense mutation, exon 3, impairs fibrinogen secretion	213
c.229-231del3ins12	V58insPLMX	Del and ins exon 3, premature stop codon	214
c.285T>A	Y76X	Nonsense mutation, exon 3	215
c.356C>G	S100X	Nonsense mutation, exon 3 escapes NMD	213
c.364+1-+4del(GTAA)		Spl. site mutation, intron 3, causes intron skipping	215,216
c.385C>T	R110X	Nonsense mutation, exon 4, escapes NMD	212
c.431-432del(AA)	K125Sfs	Fs mutation, exon 4	210
c.448C>T	Q131X	Nonsense mutation, exon 4	217
c.502C>T	R149X	Nonsense mutation, exon 4, escapes NMD	212,218
c.510+1G>T		Spl. site mutation, intron 4, causes cryptic spl. site use	215,216
15 kb del		Large deletion exons 5 and 6	219
c.541C>T	R162X	Nonsense mutation, exon 5	220
c.563-564insT	L169Ffs	Fs mutation, exon 5	220
c.607C>T	Q184X	Nonsense mutation, exon 5	221
c.609-610insTGA	L185X	Insertion, stop codon, exon 5	211
c.635T>G	L166X	Nonsense mutation, exon 5	220
c.711-712insT	K219X	Fs mutation, exon 5	222
c.743G>A	W229X	Nonsense mutation, exon 5	220
c.786-789delGAGA	E243Dfs	Fs mutation, exon 5 predicted to encode 157 aberrant aa	223
c.835delA	T260Pfs	Fs mutation, exon 5, predicted to encode 141 aberrant aa	224
c.885G>A	W276X	Nonsense mutation, exon 5	220
c.934delA	S293Afs	Fs mutation, exon 5 predicted to encode108 aberrant aa	215
c.945delT	G297Efs	Fs mutation, exon 5, predicted to encode 103 aberrant aa	215
c.946G>T	G297X	Nonsense mutation exon 5	210
c. 1001G>A	W315X	Nonsense mutation exon 5	210
c.1025delG	G323Efs	Fs mutation exon 5, predicted to encode 78 aberrant aa	220
c.1037delA	N327Tfs	Fs exon 5, predicted to encode 74 aberrant aa	220
c.1055delC	P333Lfs	Fs mutation exon 5 predicted to encode 68 aberrant aa	210
*FGB*
c.114+ 2076A>G		Mutation in intron 2 causes expression of a 50 bp cryptic exon	226
c.139C>T	R17X	Nonsense exon 2	227
c.248-249delAGinsT	K53Ifs	Fs exon 2	220
c.605T>A	L172N	Missense mutation exon 4 leads to aberrant splicing	228
c.887G>A	W266X	Nonsense mutation exon 6	214
c.958+13C>T		Splice site mutation intron 6	229
c.1148T>G	L353R	Missense exon 7 impairs secretion	230
c.1244+1G>T		Splice site mutation intron 7	229
c.1289G>A	D400D	Missense exon 8 impairs secretion	230
c.1330G>C	G414S	Missense exon 8 impairs secretion	231
c.1399T>G	W437G	Missense exon 8 impairs secretion	232
c.1400G>A	W437X	Nonsense exon 8 impairs secretion	233
*FGG*
c.78+5G>A		Spl. site mutation	234
c.98delA	N7Tfs	Fs in exon 2	215
c.124-3C>G		Spl. site mutation intron 2	215
c.307+5G>A		Spl. site mutation intron 3	220
c.400C>T	R108X	Nonsense, split between exons 4 and 5, escapes NMD	9
c.448delC	L124X	Fs exon 5 creates stop codon	5
c.666+660A>T		Intron 6 causes 75bp cryptic exon inclusion	9
c.667A>T	R197X	Nonsense exon 7	225
c.759G>T	E231X	Nonsense exon 7	9
2395G>A (5th nt of IVS-3)	77Stop,	impaired hexamer assembly	250
Del, deletion; Ins, insertion; Spl, splice; Fs, frame shift, bp, base pairs.

FIGURE 54.2 Schematic representation of fibrinogen and its conversion to fibrin. Release of FpA initiates polymerization. Each central (or E shown in red) fibrin region associates with the outer (or D) regions of two other molecules initiating a double stranded, half staggered, two molecule thick fibril, known as protofibril. As elongation progresses FpB release, significantly slower than that of FpA, is accelerated. Following FpA release, illustrated in Fig. 54.1, the αCs become untethered and dissociate. Each free αC associates with one or more counterpart(s) on neighboring fibrils (not shown) and in this manner promotes lateral assembly.

Defects in the “A” Knob

Variants include G17V (Bremen), P18L (Kanazawa and Kyoto II), R19S (Detroit), R19G (Oslo IV, Aarhus, Mannheim I, Matsumoto V, and Milano XIII), R19N (Munich I), and V20D (Canterbury). Structural data indicate that the free amino group of the “A” mimic (GPRPam) interacts directly with the side chain of residue γD364 in the “a” pocket, suggesting that the side chain is not critical.⁵⁴ Apparently, substitution of a Val for Gly17 impairs both thrombin-catalyzed fibrinopeptide A (FpA) release and polymerization since the peptide Val-Pro-Arg-Val partially inhibits fibrin polymerization.⁵⁸ Structure data also show that the positively charged side chain of R19 interacts with residues in the “a” site, such as γD330. Consequently, substitutions with Gly, Ser, or Asn will abolish these interactions. The substitution of Leu for Pro18 likely alters the structure of the “A” knob and thereby impairs the strength of the “A”:“a” interaction.
Unexpectedly, the heterozygous AαV20D substitution⁵⁹ results in absence of residues Aα1-20, thus explaining the approximately half of normal amount of released FpA. This apparently reflects a new furin-susceptible Aα20-21 bond, which enables intracellular removal of the mutant peptide.

Table 54.2 Mutations resulting in hypofibrinogenemia

*FGA*
Name, Reported Nucleotide Defect	Peptide Defect	Site	Clinical Feature(s)	Type^Reference
-1138C>T, upstream of FGA	(decreases enhancer activity)	Promoter		MS²³⁵
c.180+2T>C, spl. site		FGA intron 2		Spl²³⁶
Le Seyne, 6292T>G	AαC36G	E		MS³⁶
Marseille, 6292T>C	AαC36R	E		MS³⁶
Marseille II, 6780G>A	AαC45Y	E		MS³⁶
Cremona, 1749G>T	AαC45F	E		MS³⁸
Caracas VI	AαN80del	CC	Hem. Thr.	Del¹¹⁹
Montreal, c.1543G>A	AαD496N	αC-dom.		MS²³⁷
Grand Lyon, 5011-5012 replaced by TTGGAAATTT>Fs	Aα last 62 residues replaced by 72 new residues	αC-dom.	Hem.	Fs²⁰⁹
*FGB*
c.958+1G>A		Spl. site		Spl.⁴²
Tottori II, 3356T>G	BβY41Stop	E	Hem.	Stop²³⁸
3404A del	Bβ59PFs	E		Fs²³⁹
	BβY71X	E		NS⁷
Lyon, 4075T>A	BβM118K	CC	Hem. Misc’ge	MS¹⁶²
	BβL202Q/R47X	D/CC		MS/NS²³⁸
Paris IX, 5909A>G/IVS7-1G>C	BβY236C	D	Hem. Thr. Misc’ge	MS¹⁶³
Merivale, 6654G>A, Hannover XII, homoz.	Bβ R255H	D	Hem.	MS^40,240
Hannover XV	Bβ R255C	D		MS²⁴⁰
Nottingham II	Bβ R294G	D		MS²⁴¹
	BβD316Y	D		MS²⁴²
IVS6+1G>A, Fs	BβP319+18 residues	D		Stop²²⁹
7611 A>G	BβN320G	D		MS⁴¹
Villeurbanne II, c.1067A>G	BβY326C	D	Hem.	MS²⁴³
	BβD346Y	D		MS⁴²
Patient A, 7893 CAG>TAG	BβG393X	D	Hem.	NS⁴¹
	BβQ396X	D		NS⁴¹
	BβW402X	“b”		NS¹⁶²
c.1328A>G	BβA413S	“b”	Hem.	MS²⁴⁴
c.1331G>C	BβG414A	“b”	Hem.	MS²⁴⁴
Hannover XVIII/XX and Unicov	BβG414S	“b”		MS²⁴⁰
7969>7972, GGGG>GGG	BβG419V, Fs 419>434, 435Stop	“b”	Hem.	Stop²⁴⁵
Lyon II, 7919A>G	BβW432X	“b”		NS¹⁶²
Mumbai, homoph.	BβG434D	D		MS²⁴⁶
	BβG444S/W47X	D		MS/NS²⁴⁷
Spl. site	Bβ440-461del	D		Del²⁴⁸
	BβW470X	D	Hem.	NS²⁴⁹
	BβD496N	D		MS²⁴³
*FGG*
2525C>G, spl. site G>A	γA82G, homoz.	CC	Severe Hem. Misc’ges	MS^164,251
Poznan, c.331A>T	γ85KStop	CC	Hem.	Stop²⁵²
Matsumoto IV	γC153R	D		MS⁴⁴
Colombus. c.677G>T	γG200V	D		MS¹⁵³
Bratislava, Imp’d secr’n	γW227C	D	Hem.	MS²⁵³
Lyon IV, 5857A>C	γN230H	D		MS¹⁶²
Middlemore	γN230D	D		MS²⁵⁴
Lyon IV	γN256H	D		MS¹⁶²
Brescia	γG284R	DD	Hep. Storage	MS⁴⁷
Dorfen	γA289V	DD	Hem.	MS²⁵⁵
Hillsborough	γG309D	D		MS²⁵⁶
Manheim II	γH307Y	D		MS²⁵⁷
7590G>A	γS313N	D		MS²⁵⁸
Al du Pont, c.1018C>A	γT314P	D	Hep. storage	MS⁴⁹
	γD316Y	D		MS²⁴²
Patient B, 7611A>G	γD320G	D		MS⁴¹
Lyon III, c.1099G>A	γA341T	“a”		MS²⁴³
Tolaga Bay, c.1100C>T	γA341V	“a”		MS⁹⁶
Impaired secretion	γN345S	“a”	Hem.	MS³⁸
Angers, 7690A-7704G del	γ346-350del	“a”	Hep. Storage	Del⁵¹
Muncie, C>T	γT371I	“a”	Hem.	MS²⁵⁹
Aguadilla, C>T	γR375W	“a”	Hep. Storage	MS⁴⁸
Matsumoto VII, 7651G del	γ387Ifs>25 res>Stop	D		Fs²⁶⁰
Misc’ge, miscarriage; Spl., splice; Fs, frameshift; MS, missense; NS, nonsense; Hem., hemorrhagic; E, E region; “a”, “a” pocket; “b”, “b” pocket; D, unspecified D region site(s); Dom.: domain, CC, coiled coil.

Defects in FpA Release

Substitutions at AαR16 are most frequent with over 80 unrelated families known to date. Most are heterophenotypic and asymptomatic. They include either AαR16H or AαR16C and one AαR16S family.⁶⁰ Unique to AαR16H is the slower FpA than fibrinopeptide B (FpB) release.³² In contrast, thrombin does not cleave FpA from AαR16C fibrinogen, although it does from its modified (e.g., S-aminoethylated) isolated chains.⁶¹^,⁶² In the rare homophenotypic probands (i.e., all fibrinogen consists of abnormal homodimers) with either AαR16H³²^,⁶³ or AαR16C,⁶⁴^,⁶⁵^,⁶⁶ clotting times are prolonged by the former and infinite by the latter. In such AαR16C probands, this reflects fibrinogen incoagulability, since FpB is releasable, but no clot forms under physiologic conditions.⁶⁷ In the case of AαR16H, the reversed FpA/FpB release sequence results in clots formed when most if not all FpB and minor amounts of FpA have been released.

Substitutions of AαD7N, AαL9P, AαP11G, and AαG12V all display delayed FpA release, underscoring the importance of this region on FpA cleavage. Studies including nuclear magnetic resonance and crystallography demonstrate extensive interactions between this region and thrombin, including disruption of a critical electrostatic interaction by the AαD7N (Lille) substitution.⁶⁸ Introduction of Pro into the α-helical stretch between residues Aα7 and 10 (AαL9P, Magdeburg I) is believed to inhibit interaction with thrombin by altering conformation.⁶⁹ Similarly, the AαG12V (Rouen and Saint-Germain) substitution is predicted to disrupt the Aα1-12 β turn and slightly displace Gly13 and Gly14.⁷⁰ The Saint-Germain report also describes delayed FpB release.⁷¹ In addition, AαE11G (Mitaka II) destabilizes the β turn and disrupts the salt bridge between fibrinogen AαE11 and R173 of thrombin.⁷²

Defects in FpB Release and B Knob

Only heterophenotypic Cys substitutions were identified at or near the Bβ14 thrombin cleavage site (Table 54.3). Cleavage of FpB exposes the N-terminal β chain site, “B” knob, which binds noncovalently to a complementary polymerization “b” pocket
in the C-terminal region in the β chain (β397-432) of another fibrin(ogen) molecule. This interaction is modeled in the crystal structures obtained in the presence of GHRPam (see previous reviews⁷^,⁸). Only one substitution has been found in the “B” knob: heterophenotypic BβG15C (Ise, Fukuoka II, Kosai, Ogasa, and Hamamatsu II). Functional studies showed normal FpA release, approximately 50% FpB release, impaired thrombin- and reptilase-catalyzed polymerization, and impaired desA- and desAB-fibrin monomer repolymerization.⁷³^,⁷⁴ In addition to impairing the function of “B” knob, these results suggest they impair that of the “A” knob whose second part⁸ resides within β.15-42 The sulfhydryl group of each varied. Some formed complexes with albumin attributable to disulfide links via the albumin’s solitary-free C34 sulfhydryl,⁷⁵ some formed fibrinogen-fibrinogen dimers, and some were free sulfhydryls. Three were asymptomatic and two were associated with thrombosis.

Among eight BβR14C unrelated probands (including those in the online registry, vide supra), six were associated with thrombotic events,⁷⁶^,⁷⁷ occurring at a young age in at least four, and in several probands in one family (Table 54.3). This association, however, has not occurred in every affected family and remains unclear. There was one proband with hemorrhage and one asymptomatic.⁷⁸ Release of FpA was normal and release of FpB was half-normal. Reptilase-induced polymerization (Seattle) was also impaired, suggesting that the mutation itself impairs polymerization.⁷⁸ Studies on fibrinogen Ijmuiden identified molecular species linked to albumin (40%), in free sulfhydryl form (36%), and as fibrinogen-fibrinogen dimers (30%).⁷⁷ BβR44C (Nijmegen, heterophenotypic) impaired fibrin polymerization. Its association with thrombosis contrasts with the same mutation in two unrelated and asymptomatic families (author’s unpublished data).

Defects in Thrombin Binding

Three different Bβ chain substitutions resulted in delayed fibrinopeptide release linked to impaired thrombin binding. One was the homophenotypic BβA68T (Naples) substitution that impaired thrombin binding (<10%) to fibrin and markedly delayed FpA and FpB release.⁷⁹ Crystallographic data suggest that this substitution impairs interactions between fibrinogen and thrombin exosite I.⁸⁰ Other studies indicate that binding of thrombin to low-affinity sites (located in the E region) is abolished and high-affinity binding to the

chain is impaired.⁸¹ The second is a heterophenotypic fibrinogen (New York I) with a large deletion of residues Bβ9-72, encoded by FGB exon 2.⁸² Its delayed FpA and FpB release is also attributable to diminished thrombin binding. A third substitution, a heterophenotypic BβA61K (Miami),⁸³ is associated with repeated spontaneous abortions and impaired polymerization. A reptilase-incoagulable subfraction (60% of plasma fibrinogen) displayed thrombin binding <10% of normal controls (author’s unpublished data).

Defects that Impair Fibrin Polymerization

The intermolecular D:E interaction results in half-staggered, double-stranded fibrils (FIGURE 54.2) and lateral association of fibrils with fibers. More specific interactions have been elucidated in x-ray crystallographic structures of fibrinogen fragments D and FXIIIa-cross-linked fragment D-D in the presence of the peptides GPRPam and GHRPam, which mimic the N-terminal regions of the α and β chains of fibrin, respectively.⁵⁴^,⁵⁶^,⁵⁷^,⁸⁴ Identification of specific amino acids in these structures and their substituted counterparts caused by mutations enabled a correlation of corresponding biochemical data with the three-dimensional structure. Also, binding of calcium to high-affinity sites γAsp318, Asp320, Phe322, and Gly324⁸⁴ is impaired by mutations in this stretch.

Defects within the “a” Pocket

This pocket encompasses γ chain residues 337 to 379. Crystallographic studies showed that Q329 shifts on GPRPam binding; the substitution γQ329R (Nagoya) may disrupt this shift, thereby impairing polymerization.⁸⁵ Substitutions γD330V, Ales⁸⁶ and Milano I,⁸⁷ and D330Y, Kyoto III⁸⁸ disrupt ionic interactions with AαR19. Biochemical analyses of these variants demonstrate that the AαR19 residue is critical for polymerization.⁸⁶^,⁸⁸ Similarly, substitutions γD364 to His and Val, Matsumoto I⁸⁹ and Melun⁹⁰ respectively, disrupt ionic interactions with the free amino group of the “A” knob mimic, and both substitutions impair polymerization. A γ-chain insertion of 15 residues between γQ350 and γG351, with a concomitant G351S change (Paris I), was first noted in the protein⁹¹^,⁹² and later defined by genetic analyses.⁹³ The inserted segment has two Cys residues that likely form a disulfide bond, which alter conformation around residues γ350 and γ351. These alterations can account for the impaired polymerization, factor XIIIa cross-linking, and platelet binding.⁹⁴ A study of three γ364 substitutions, Ala, His, or Val, disclosed coagulability with normal FpA release but absence of “A”: “a” interactions, implying that “B”: “b” interactions enabled polymerization.⁹⁵ This is in general agreement with the polymerization behavior of homophenotypic AαR16H (Kingsport).³² Two polymerization-impairing mutations near the “a” pocket resulted in novel glycosylation γA327T (Tokyo V) and γK380N (Kaiserslautern I). Impaired polymerization by the latter with a new glycosylation site at N380 was corrected by desialylation, suggesting the extra sialylation impaired polymerization. Substitutions γA341D and γA341V⁹⁶^,⁹⁷ resulted in impaired “A”:“a” interactions and impaired calcium binding,⁹⁸ implying that this residue is required for both functions. Two novel Cys substitutions within the “a” pocket were covalently linked to albumin, γY354C⁹⁹ (Homburg VII), and γS358C¹⁰⁰ (Milano VII). Both impaired polymerization, and this was not corrected by removal of albumin from Milano VII, suggesting it was attributable to γ358C per se.

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