SummaryPooled plasma from 40 patients with severe disseminated intravascuiar coagulation (DIC) secondary to septic conditions was subjected to gel permeation chromatography on Sephacryl S-500 HR after sample pretreatment with KSCN for dissociation of non-covalent fibrin complexes. Fibrin antigen in eluates was detected by an array of ELISA tests, using two monoclonal antibodies against fibrin degradation product D-dimer, a monoclonal antibody against an epitope generated by plasmin cleavage of the D-domain, and an antibody against the neo-N- terminus of the α-chain of fibrin exposed by cleavage of fibrinopeptide A. Tag antibodies were a polyclonal antibody against the fibrinogen/ fibrin D-domain, a POD-conjugated version of the monoclonal antibody against fibrin α-chain neo-N-terminus, and a polyclonal antibody against fibrinopeptide A. Most fibrin-related material present in the pooled DIC plasma was of higher molecular mass than fibrinogen. Fibrin polymers were reactive with antibodies against D-dimer, plasmin cleaved D-domain, and fibrin α-chain neo-N-terminus. Part of the polymers reacted with antibodies against fibrinopeptide A, indicating presence of fibrinogen or desA-fibrin monomer within the covalently linked complex. In conclusion, the primary analytes detected by monoclonal antibodies for D-dimer, plasmin-specific epitopes of fibrin degradation products, as well as sites exposed by fibrinopeptide cleavage in plasma from patients with disseminated intravascuiar coagulation are high molecular weight factor XIIIa-crosslinked fibrin complexes, containing plasmin-cleaved D-domains, intact fibrin monomer units, and fibrinogen or desA-fibrin monomer.
SummaryVarious assays have been developed for quantitation of soluble fibrin or fibrin monomer in clinical plasma samples, since this parameter directly reflects in vivo thrombin action on fibrinogen. Using plasma samples from healthy blood donors, patients with cerebral ischemic insult, patients with septicemia, and patients with venous thrombosis, we compared two immunologic tests using monoclonal antibodies against fibrin-specific neo-epitopes, and two functional tests based on the cofactor activity of soluble fibrin complexes in tPA-induced plasminogen activation. Test A (Enzymun®-Test FM) showed the best discriminating power among normal range and pathological samples. Test B (Fibrinostika® soluble fibrin) clearly separated normal range from pathological samples, but failed to discriminate among samples from patients with low grade coagulation activation in septicemia, and massive activation in venous thrombosis. Functional test C (Fibrin monomer test Behring) displayed good discriminating power between normal and pathological range samples, and correlated with test A (r = 0.61), whereas assay D (Coa-Set® Fibrin monomer) showed little discriminating power at values below 10 μg/ml and little correlation with other assays. Standardization of assays will require further characterization of analytes detected.
SummaryHuman plasma fibrinogen is heterogeneous in SDS-polyacrylamide gel electrophoresis and other methods for separation of proteins by molecular size. A high molecular weight fraction (HMW-fibrinogen, 340 kD) contributes approximately 50% of total fibrinogen antigen. Low molecular weight fibrinogen (LMW-fibrinogen, 300 kD) adds another 40%. The residual amount is LMW’-fibrinogen with a molecular weight of 280 kD, and a small amount of very high molecular weight fibrinogen (Fib420), the product of alternative splicing of the Aa-chain genetic information, resulting in an extended A?-chain C-terminus. Fibrinogen was detected by specific immunostaining of nonreduced SDS-PAGE gel immunoblots with antibodies against fibrinopeptide A. Using densitometric scans of the immunoblots we found a ratio of HMW-, LMW- and LMW’-fibrinogen in a patient with homozygous plasminogen deficiency that was similar to the ratio found in immunoblots of plasma from healthy blood donors. Treatment with plasminogen concentrate resulted in a slight decrease of the proportion of HMW-fibrinogen, followed by an increase to 78%. The LMW’- fibrinogen band gained intensity initially, increasing to 11.9% of fibrinogen antigen 6 h after starting plasminogen infusion, but then dropped to levels below detection limit of the immunoblotting assay. LMW-fibrinogen remained constant during the initial 72 h of plasminogen treatment, then dropping to values in the range of 22-25% afterwards. The proportion of HMW-, LMW-, and LMW’-fibrinogen.again reached the initial levels two weeks after starting treatment with plasminogen concentrate. We conclude that plasminogen is not involved in the limited proteolysis leading to formation of LMW-fibrinogen and LMW’-fibrinogen in the absence of a generalized fibrinolytic condition. Fibrinolytic activation may lead to the formation of fibrinogen degradation product X, which appears in a similar position as LMW’- fibrinogen in SDS-PAGE.
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