The Polymerization of Fibrin Prepared from Fibrinogen Haifa (γ275Arg→His)

Abstract

SummaryFibrinogen Haifa is a congenital heterozygous fibinogen variant (γ275 Arg→His) characterized by prolonged thrombin and reptilase times and normal fibrinopeptide (FPA, FPB) release. We compared the polymerization rate (by turbidity measurements at 350 nm) and the ultrastructure of Haifa α-, β-, and α, β-fibrin with that of normal. Haifa α, β-fibrin polymerized less rapidly than did normal and formed a highly branched matrix with a smaller mean fiber diameter; this network closely resembled that of normal α, β-fibrin with EDTA added. In the presence of CaCl2 (1 to 10 mM), Haifa α, β-fibrin polymerized more rapidly than in buffer alone and possessed a matrix structure closely resembling that of normal fibrin. From these observations it appears that the functional defect in Haifa fibrin can be related to the inability of the abnormal molecule to effectively utilize available calcium. The polymerization profile of Haifa α-fibrin differed only modestly from that of normal α-fibrin, whereas that of Haifa β-fibrin was markedly impaired. This finding plus similarities in the ultrastructure of Haifa and normal α-fibrin specimens suggests that the defective γ chain structure of Haifa fibrinogen results in greater impairment of the carboxy terminal “b” polymerization domain reacting with the site exposed by cleavage of FPB (“B” site) than it does that of the carboxy terminal “a” domain reacting with the site exposed by cleavage of FPA (“A” site). Whether this effect is due to absolute differences in the degree of impairment of these two types of polymerization sites, or whether proper utilization of the “B” to “b” site is dependent upon participation of the “A” to “a” site remains to be determined.

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

“…The Tokyo II fibrin network is more highly branched than its normal counterpart (Fig. 2), an observation that is very similar to those we have made on fibrinogens having a mutation at the same site-Haifa (y275 arg--his) (31) and Morioka (y275 arg-.cys) (32). The likely explanation for increased branching is that impairment of intermolecular D:D interactions permits fibrin molecules to extend outward from linearly propagating fibril strands more frequently, thus favoring formation of greater numbers of branch points (25).…”

Localization of an Effective Fibrin β-Chain Polymerization Site: Implications for the Polymerization Mechanism

“…Although the turbidity assay has been useful for identifying small peptide polymerization sites (Laudano & Doolittle, 1978;Hasegawa & Sasaki, 1990), a markedly lower A 350 turbidity was observed for desBB-than for desAA-fibrin polymers (Mosesson et al, 1987;Lee et al, 1991), in spite of the indistinguishable desBB-, desAA-, and desAABB-fibrin fibers found in electron microscopy and X-ray studies (Weisel, 1986;Voter et al, 1986). Paradoxically, the A 350 turbidity of fibrin clots was decreased by 40% when fibrin monomers were repolymerized in the presence of calcium (from 1.066 to 0.641; Siebenlist et al, 1990;Maekawa et al, 1992) but increased when fibrinogen was clotted by thrombin, reptilase, or copperhead venom in the presence of calcium (Siebenlist et al, 1989;Dang et al, 1989b;Lee et al, 1991).…”

Fibrinogen Kurashiki (γ G268E) Fibrin Network Structure

“…The unusual appearance of this matrix is consistent with previous SEM findings on fibrin formed from dysfibrinogens with defects at the D:D association site, such as fibrinogen Tokyo II (yR275C) 2 , that showed a defective clot structure characterized by an extensively branched and contoured, relatively thick fiber network. Ultrastructural studies on fibrin from other dysfibrinogenemias with y275 defects [e.g., fibrinogens Haifa (yR275H) 4 , Morioka (y R275C) 5 and Cedar Rapids yR275C) 6 ] also revealed abnormally branched thick fiber clot structures, clearly indicating that self-association defects at the D:D site result in the same type of aberrant fibrin assembly.…”