SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Specific binding of purified histidine rich glycoprotein (HRGP) to human platelets stimulated with either bisdiazoniumbenzidine-crosslinked immunoglobulin G (BDB-IgG), with thrombin or with collagen was dose- and divalent cation dependent. A 5-10-fold increase of platelet bound 125I-HRGP was obtained when 0.5-0.8 x 10(9) platelets/ml were activated with 100 micrograms BDB-IgG/ml, 0.1 U thrombin/ml or 15 micrograms collagen/ml. At maximal binding tested 16,000 molecules of HRGP became bound per platelet, but saturation was not achieved. Such platelet inhibitors as acetylsalicylic acid, prostaglandin E1 and cytochalasin B reduced the capacity of platelets to bind ligand, and by kinetic experiments involving enzymatic digestion of radiolabelled bound HRGP the ligand revealed to remain surface bound rather than being taken up to inner parts of the cell.

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Inherited hypodysfibrinogenemia (fibrinogen Bern I) was found in four members (two generations) of a family with no haemorrhagic or thrombotic history. Fibrin aggregation curves (350 nm, 37°C) with patient plasma or purified fibrinogen Bern I, after addition of thrombin, were normal at high calcium concentrations (5mM) but delayed at lower calcium concentrations (≤0.lmM). The release of fibrinopeptide A was normal. Whereas the polypeptide chains of fibrinogen Bern I were indistinguishable from normal fibrinogen by SDS-gel-electrophoresis, an abnormal γ-chain with a decreased negative charge was found by isoelectric focussing.Plasmic degradation o| normal fibrinogen, in the presence of calcium (≥ImM), results in only one terminal D fragment which is stabilized by calcium against further degradation of γ-chains. In contrast, degradation of fibrinogen Bern I, under the same conditions, yielded at least two additional smaller D fragments. In conclusion, fibrinogen Bern I is characterized by defective calcium binding in the D domain of the γ-chain.