Mann, K G

Abstract Immunization of goats and mules with human thrombin resulted in an antiserum that reacted only weakly with the parent molecule, prothrombin. Some of the antibodies in this antiserum showed a greater affinity for thrombin complexed to its naturally occurring inhibitor, antithrombin-III, than for active thrombin. An antiserum against the human thrombin inhibitor, antithrombin-III, produced 2 precipitin lines against human serum but only 1 against plasma. The 2nd line in serum was shown to represent precipitation of a complex of thrombin with antithrombin-III. The neoantigens appearing in antithrombin-III after complex formation were also present in complexes prepared with purified clotting factor Xa and antithrombin-III. Since purified host (mule) thrombin was also capable of causing formation of the neoantigenic sites when complexed to human antithrombin-III, it seems likely that these determinants result from interaction in the host between the immunogens (either human thrombin or antithrombin) and the appropriate interacting host protein (mule antithrombin-III or thrombin, respectively). Studies by radioimmunoassay showed that the antibodies formed are not completely specific for the neoantigens since they also react to a lesser extent with the free proteins.

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of nornar thrombin (T). The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. when incubated with antithrombin III, both T ad TQ fom inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinquished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 × 10-5M and kcat= 6.93 compared to Km = 4.0 × 10-5M and kcat= 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for hydrolysis of the active site titrant 4-methykl-umbelliferyl-p-guanidinobenzoate

Bovine Factor V was incubated briefly with bovine β- thrombin, causing partial conversion to Factor Va. QAE- cellulose chromatography in Ca++ of the partially activated material yielded 3 fractions. Electrophoresis in DodSO4 indicated primarily starting material (Mr=330,000) in the first fraction; two peptides of Mr=150,000 and 200,000 in the second; and two peptides of Mr=200,000 and 90,000 in the third. The first of these latter fractions was active only after further incubation with thrombin, while the other was active without further thrombin catalyzed activation. After thrombin treatment both fractions had specific activities equal to that of fully activated, unfractionated Factor V. Incubation of either of these fractions with EDTA caused complete loss of activity and allowed isolation of the constituent polypeptides by further chromatography on QAE- cellulose in EDTA. The NH2-termini of the 150,000 and 90.000 apparent Mr peptides had sequences identical to that of Factor V. The 200,000 apparent Mr peptides of the two fractions obtained by chromatography in Ca++ had NH2- terminal sequences identical to each other but different from that of Factor V. The summed amino acid compositions of the Mr=200,000 and Mr=150,000 were equivalent to that of Factor V. Electrophoretic analysis indicated that thrombin converts the Mr=150,000 peptide to endproducts of Mr=90,000 and 71,000, and the Mr=200,000 peptide to endproducts of 83.000 and 31,000 apparent Mr. Recalcification of the individual isolated intermediates, followed by incubation with thrombin did not yield cofactor activity. Recombina- ion of the recalcified intermediates followed by incubation with thrombin, however, generated samples with activities approaching that of unfractionated, activated Factor V. These data indicate that the intermediates of Mr=150,000 and Mr=200,000 arise by a single cleavage of Factor V, and that Factor Va comprises peptides derived from both of these intermediates.

It has generally been assumed that the thrombogenicity of PCC relates to their invariable contamination by activated products of their component clotting factors. In earlier studies we demonstrated a lack of correlation between the activated clotting factor content ᶓ in vivo thrombogenicity of some PCC. In contrast, in vitro measurement of coagulant active PL content appeared to be a more reliable determinant of subsequent in vivo activity. In recent studies we have confirmed the presence of PL of known coagulant activity in PCC ᶓ studied its activity both in vitro ᶓ in vivo after its extraction from the parent PCC.PCCs were obtained from various agencies or prepared in our laboratory to a standard protocol either from fresh frozen plasma or plasma obtained from outdated platelet concentrates. Each was assayed for Xa (S-2222) ᶓ PL (thrombin generation assay with DAPA) ᶓ an index of thrombogenicity in vivo determined in a stasis model in rabbits. There was a significant correlation between the latter ᶓ PL (r=0.8727 p<0.01) but not Xa. Thin layer chromatography of an ethanol ether extract of the most thrombogenic PCC showed the presence of PL known to be coagulant active including Phospho- tidylserine ᶓ -choline. This extract retained full PL replacing activity in the DAPA assay in vitro but was non- thrombogenic in vivo. When, however, a highly purified preparation of Xa was added, in the same relative proportions as in the parent PCC, thrombogenicity in vivo was restored.It is concluded that the content of coagulant active PL is a critical determinant of the potential thrombogenicity of PCC in vivo. It appears to act in concert with Xa ᶓ conceivably, other serine proteases perhaps by protecting them from inactivation by AT III. This may explain why relatively “non-activated” PCCs may still be associated with thromboembolic complications.

The peptides produced during the thrombin (IIa) catalyzed activation of human Factor V (HFV) were investigated using a murine monoclonal antibody. This antibody (HFV-1) binds HFV and activated HFV (HFVa) and releases them at high ionic strength. HFV-1 coupled to sepharose was used to purify the single chain, 330,000 apparent MW procofactor molecule. Gel electrophoresis in SDS indicated that IIa- mediated activation proceeded with the appearance of intermediates with apparent MW of 270K, 205K and 150K and resulted in at least 3 end products with apparent MW of 110K, 93K and 70K. Aliquots of partially and fully activated HFV were chromatographed on the HFV-1 affinity resin. The peptides not associated with HFVa were obtained by washing the column with 20 mM imidazole, 0.154 M NaCl, 5 mM CaCl2, pH 6.5; the peptides noncovalently bound to the epitope-containing peptides were obtained by elution in the above buffer made 5 mM EDTA in place of Ca++ and the epitope-containing peptides were eluted in buffer made 1.2 M in NaCl. The 150K intermediate was released in EDTA and the 205K and 270K peptides were eluted in high salt. Of the end products of activaion, the 110K peptide was not retained by the coliman, the 93K peptide was released in EDTA, and the 70K peptide was released only at high ionic strength. These data indicate that the 270K and 205K intermediates, and the 70K end product share the antigenic site involved in the interaction with the monoclonal antibody. In addition a Ca++- dependent interaction between the epitope-containing domain and the 150K intermediate and 93K end product is suggested. From this data and by analogy with the bovine protein, we suggest that HFVa is a Ca++-dependent multiple subunit protein consisting minimally of two peptides of apparent MW of 93,000 and 70,000.