The relationship of clinical bleeding tendency and factor XI antigen (XI:Ag) in factor XI deficiency was studied in 78 members of 25 factor XI-deficient kindreds. Factor XI:Ag was measured in a competitive radioimmunoassay, using monospecific, heterologous anti-factor XI antibody. 125I-labeled factor XI, and staphylococcal protein A as the precipitating agent. Deficiency of factor XI clotting activity (XI:C), less than 0.62 U/mL, occurred in 48 individuals, 22 of whom experienced postoperative or posttraumatic bleeding: Their mean factor XI:C was 0.21 +/- 0.04 U/mL (SEM), and factor XI:Ag was 0.23 +/- 0.04 U/mL. The remaining 26 had no clinical bleeding, many despite surgical challenge: Their mean factor XI:C was 0.30 +/- 0.04 U/mL, and factor XI:Ag was 0.34 +/- 0.05 U/mL. In all, 13 kindreds had between 1 and 11 members with bleeding; the other 12 had none with deficient hemostasis. Two heterozygous factor XI-deficient individuals appeared to be positive for cross-reacting material (CRM+). The slope of the regression line for factor XI:C and factor XI:Ag data points in the 78 individuals tested did not differ from control, and all points fell within 95% confidence limits derived from control. In conclusion, bleeding tendency appears to be consistent within a given kindred and is not determined exclusively by factor XI:C or factor XI:Ag levels.

Abstract. Binding of '25I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 370C. Factor XIa binding was specific: 50-to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversible and was saturable at concentrations of added Factor XIa of 0.2-0.4 ug/ml (1.25-2.5 MM).

Since optimal rates of factor IX activation by factor XIa require the presence of calcium ions and the heavy chain of the enzyme as well as the active-site-containing light chain, we have studied the effects of calcium ions and the heavy chain on the reaction kinetics. Whereas the amidolytic activities of factor XIa and of its active-site-containing light chain were almost indistinguishable, the two enzymes behaved quite differently when factor IX was the substrate. Factor XIa was 100-fold more potent in the presence of Ca2+ than in its absence. On the contrary, the presence or absence of Ca2+ made very little difference in the case of the isolated light chain of factor XIa. Moreover, the enzymatic activity of the light chain was almost identical with that of intact factor XIa when Ca2+ was absent. Using an optimal concentration of Ca2+, we studied the activation in the presence of various concentrations of two monoclonal antibodies, one (5F4) directed against the light chain of factor XIa and the other (3C1) against its heavy chain. Analysis of 1/V vs. 1/S plots showed that whereas inhibition by 5F4 was noncompetitive, 3C1 neutralized the enzyme in a classical competitive fashion. We conclude that in the calcium-dependent activation of factor IX by factor XIa the heavy chain of the enzyme is involved in the binding of the substrate and this is essential for optimal reaction rates.

We have previously demonstrated the presence of a binding site for high-molecular-mass kininogen (HK), spanning residues Val59-Lys83, in the first Apple (A1) domain in the heavy-chain region of factor XI. We have now prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) constructs to identify the specific amino acid residues that constitute the HK-binding site. Expression of the A1 domain (Glu1-Ser90) was achieved in a bacterial expression system following PCR amplification of the A1 domain from factor XI cDNA and ligation into an expression plasmid. The rA1 inhibited factor XI binding to HK [Ki approximately (2-3) x 10(-7) M] in a manner indistinguishable from purified factor XI, indicating that all the information necessary for binding HK is contained within the A1 domain. To identify specific amino acid residues involved in binding HK, conformationally constrained peptides were synthesized containing conservative amino acid substitutions at residues suspected to contain side chains involved in binding, including Val64-->Ala, Glu66-->Ala, Arg73-->Ala and Ile77-->Ala. Because normal results were obtained with all peptides with the exception of Val64-->Ala and Ile77-->Ala, which failed to compete normally with factor XI for binding to HK, we prepared two mutant rA1 domains (Val64-->Ala and Ile77-->Ala) by PCR-based site-directed mutagenesis, both of which exhibited diminished capacity to inhibit factor XI binding to HK. Competition studies with prekallikrein (PK) and a PK-dependent synthetic peptide suggested that PK and factor XI have a common surface in the A1 domain for binding HK of which Val64 is a part. We conclude that the binding of factor XI to HK is mediated at least in part by Val64 and Ile77 in the A1 domain of factor XI.

We have studied the complex interrelationships between platelets, Factor XIa, a1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XMa, and to protect Factor XIa from inactivation in the presence of a1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor Ma did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of a1-protease inhibitor by platelets. The formation of a complex between a1-protease inhibitor and the active-site-containing light chain of Factor XMa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug.