Activated platelets expose a specific, reversible high affinity (Kdapp congruent to 10 nM) binding site (n congruent to 1500 sites/platelet) for factor XI that requires the presence of high molecular weight kininogen (HK) and ZnCl2 (Greengard, J. S., Heeb, M. J., Ersdal, E., Walsh, P. N., and Griffin, J. H. (1986) Biochemistry 25, 3884-3890). Synthetic, conformationally constrained peptides from four tandem repeat (Apple) domains were tested for their capacity to inhibit 125I-factor XI binding to platelets. A peptide from the Apple 3 (A3) domain (Asn235-Arg266) inhibits factor XI binding to platelets in the presence of HK (42 nM), CaCl2 (2 mM), and ZnCl2 (25 microM), with a Ki congruent to 10 nM which is identical to the Kd for factor XI binding to platelets. A peptide from the A1 domain (Phe56-Ser86) partially inhibits factor XI binding to platelets (Ki congruent to 6 microM) by inhibiting factor XI binding to HK, whereas peptides from the A2 and A4 domains have no effect. Using computer modeling for rational design, conformationally constrained peptides were synthesized (Pro229-Gln233, Thr241-Leu246, and Ser248-Ser261) each of which acted alone and synergistically when added together to inhibit factor XI binding to platelets. Finally, the 125I-labeled A3 domain peptide (Asn235-Arg266) was found to bind to thrombin-activated platelets in a specific, reversible, and saturable manner. Thus, the sequence of amino acids Asn235-Arg266 of the A3 domain of factor XI comprises a contact surface for interaction with a platelet receptor.

To study the physiological significance of thrombin as an initiator of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as a surface for thrombin-catalyzed factor XI activation. Activated gel-filtered platelets promoted factor XI activation by thrombin at initial rates 2-5-fold greater than dextran sulfate in the presence of high molecular weight kininogen (HK, 45 nM), ZnCl2 (25 microM), and CaCl2 (2 mM), conditions optimal for factor XI binding to platelets. Physiological concentrations of HK (636 nM) inhibited factor XI activation by thrombin in a concentration-dependent manner, and this inhibition was reversed by prothrombin (1-3 microM) and by prothrombin fragment 1.2 (PF1.2), but not by prothrombin fragment 1 (PF1). Since prothrombin and PF1.2 (but not PF1) also displaced HK from its binding site on the Apple 1 domain of factor XI, we conclude that the Kringle II domain of prothrombin competes with HK for binding to the Apple 1 domain of factor XI. Prothrombin (1-3 microM) and PF1.2 (but not PF1) in the presence of CaCl2 (2 mM) were able to replace HK (45 nM) in the presence of ZnCl2 (25 microM) as a cofactor for the specific, reversible, high-affinity (Kd approximately 25 nM) binding of factor XI to 947 +/- 150 sites per platelet. This binding is mediated by residues Asn 235-Arg 266 in the Apple 3 domain since a conformationally constrained, synthetic peptide analogue of this sequence inhibits both factor XI binding to activated platelets and platelet-mediated, thrombin-catalyzed factor XI activation in the presence of prothrombin and CaCl2. Finally, prothrombin (1.2 microM) and CaCl2 (2 mM) could substitute for HK (45 nM) and ZnCl2 (25 microM) in promoting optimal rates of thrombin-catalyzed factor XI activation on the platelet surface, thereby initiating the intrinsic coagulation pathway by mechanisms completely independent of the contact phase proteins, factor XII, HK, and prekallikrein.

Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Factor XI (FXI) 1 is a zymogen that circulates in plasma in a non-covalent complex with high molecular weight kininogen (HMWK) (1, 2) and participates in the contact phase of blood coagulation (3). The active enzyme, activated factor XI (FXIa), is a trypsin-like serine protease that is generated when FXI is cleaved by FXIIa (3), thrombin, or FXIa (4, 5) at an internal Arg 369 -Ile 370 bond (2). Upon activation, FXIa is capable of converting FIX into its active form, FIXa (2, 6). FIXa, in the presence of FVIII and platelets, can activate FX (7,8), resulting in the generation of thrombin and, subsequently, a fibrin clot.The structure of FXI is unique among the plasma coagulation enzymes (9, 10), since it exists as a homodimer consisting of two subunits each of which contains 607 amino acids. During proteolytic activation, each of these subunits is cleaved to generate a heavy chain of 369 amino acids and a light chain or catalytic domain of 238 amino acids. The heavy chain of FXI provides binding surfaces for several blood coagulation proteins and is organized into four tandem repeat Apple domains designated A1, A2, A3, and A4 (9, 10). Each of these four domains contains 90 to 91 amino acids, the sequences of which are 23-34% identical. The A1 domain provides a binding site for HMWK (11), a protein that promotes the activation of FXI by FXIIa (12). Both the A2 domain (13) and the A3 domain (14) have been implicated in the binding of FXIa to its substrate, FIX. The A4 domain contains Cys 321 that is responsible for the dimerization of FXI (15,16). In addition, the A4 domain binds to FXIIa (10).Examination of the cellular site of FXI activation by FXIIa has resulted in evidence that platelets promote the proteolytic activation of FXI by FXIIa (17) and that FXI, in the presence of zinc ions, calcium ions, and HMWK, binds to activated platelets in a specific, reversible, and saturable manner with a dissociation constant (K d ) of ϳ10 nM (18). Previous work from this laboratory showed that a synthetic peptide (Asn 235 -Arg 266 ) from the A3 domain inhibited 125 I-FXI binding to platelets (inhibition constant (K i ) ϭ 10 nM) in the presence of HMWK, ZnCl 2 , and CaCl 2 (19,20). Hence, these studies indicate that the A3 domain mediates FXI binding to platelets.A second possible site of FXI activation is the endothelial cell surface. Berrettini et al. (21) demonstrated that FXI binds to endothelial cells in the presence of HMWK, CaCl 2 , and ZnCl 2 with a K d(app) ϳ4.5 nM, whereas FXIa binds with higher affinity (K d(app) ϳ1.5 nM). The binding of FXI to the endothelial cell surface may be of functional significance in localizing coagulation to the site of vascular injury since activation of FXI can occur on the endothelial cell surface (21), where proteoglycans such as heparan sulfate are known to be present. Both Naito and Fujikawa (4) and Gailani an...

Factor XI binds to high affinity sites on the surface of stimulated platelets where it is efficiently activated by thrombin. Here, we provide evidence that the factor XI binding site on platelets is in the glycoprotein (GP) Ib␣ subunit of the GP Ib-IX-V complex as follows. 1) Bernard-Soulier platelets, lacking the complex, are deficient in factor XI binding; 2) two GP Ib␣ ligands, SZ-2 (a monoclonal antibody) and bovine von Willebrand factor, inhibit factor XI binding to platelets; 3) by surface plasmon resonance, factor XI bound specifically to glycocalicin (the extracellular domain of GP Ib␣) in Zn 2؉ -dependent fashion (K d app ϳ 52 nM). We then investigated whether glycocalicin could promote factor XI activation by thrombin, another GP Ib␣ ligand. In the presence of high molecular weight kininogen (45 nM), Zn 2؉ and Ca 2؉ions, thrombin activated factor XI in the presence of glycocalicin at rates comparable with those seen in the presence of dextran sulfate (1 g/ml). With higher high molecular weight kininogen concentrations (360 nM), the rate of thrombin-catalyzed factor XI activation in the presence of glycocalicin was comparable with that on activated platelets. Thus, factor XI binds to the GP Ib-IX-V complex, promoting its activation by thrombin.Coagulation factor XI (FXI) 1 is a disulfide-linked homodimer that can be bound to activated platelets and subsequently activated by thrombin, FXIIa or FXIa (1-10). In plasma, FXI circulates as a complex with high molecular weight kininogen (HK) (1-7). In the presence of HK or prothrombin, FXI binds specifically and reversibly to high affinity sites on the surface of activated human platelets in the presence of zinc and calcium ions (8,9). We have reported that activated platelets promote FXI activation by thrombin in the presence of HK or prothrombin at optimal rates, thereby initiating intrinsic coagulation independent of contact proteins (8).The primary structure of FXI reveals the presence of four repeated Apple domains (A1-A4) within the heavy chain followed by a light chain containing the serine protease catalytic domain (11). Our laboratory has demonstrated that the A3 domain of FXI is essential for the binding of FXI to platelets as both A3 domain peptides and a recombinant A3 domain bind specifically and saturably to the platelet surface (10,12). However, the platelet receptor(s) responsible for this interaction has not been identified.One potential receptor was suggested by our earlier work (13) with platelets from patients with the congenital bleeding disorder Bernard-Soulier syndrome (14,15). This disorder results from a deficiency or functional defects of the platelet glycoprotein (GP) Ib-IX-V complex, a large plasma membrane complex comprising four polypeptide chains: GP Ib␣, GP Ib␤, GP IX, and GP V, arranged in a stoichiometry of 2:2:2:1, respectively (16 -18). Approximately 25,000 copies of the first three peptides reside in the platelet surface along with half as many copies of GP V. The GP Ib-IX-V complex is the receptor responsible for adhering...

To study the pathways for initiation of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as surfaces for factor XI activation by factor XIIa, factor XIa, or thrombin. Activated gelfiltered platelets promoted the activation of factor XI (60 nM) by thrombin (0.02-10 nM, EC 50 ϳ100 pM, threshold concentration ϳ10 pM) at initial rates 2-to 3-fold greater than those obtained with dextran sulfate in the presence of either high molecular weight kininogen (45 nM) and ZnCl 2 (25 M) or prothrombin (1.2 M) and CaCl 2 (2 mM). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nM⅐min ؊1 with thrombin, 6 nM⅐min ؊1 with factor XIIa and 2 nM⅐min ؊1 with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nM) were 24 -167 (mean ‫؍‬ 86) min ؊1 for thrombin, 4.6 -50 (mean ‫؍‬ 21) min ؊1 for factor XIIa, and 1.3-14 (mean ‫؍‬ 8) min ؊1 for factor XIa. A physiological concentration of fibrinogen (9.0 M) inhibited factor XI activation by thrombin (but not by factor XIIa) in the presence of dextran sulfate but not in the presence of gel-filtered platelets. Compared with factors XIIa and XIa, thrombin is the preferred factor XI activator, and activated platelets are a relevant physiological surface for thrombinmediated initiation of intrinsic coagulation in vivo.Human coagulation factor XI is a disulfide-linked homodimer consisting of two identical polypeptide chains each containing 607 amino acids. Factor XI is present in human plasma as a zymogen that requires proteolytic activation to develop serine protease activity (1-5). It circulates in human plasma in a noncovalent complex with high molecular weight kininogen (HK) 1 (6) and can be activated by three biologically relevant proteases: factor XIIa, factor XIa, and thrombin (1,7,8). The primary structure of factor XI has been determined (9, 10), including the identification of four tandem repeat sequences, designated Apple (A1, A2, A3, and A4) domains in the heavy chain region of factor XI. Binding sites (11-15) for thrombin, the Kringle 2 domain of prothrombin, and HK are present in the A1 domain, whereas both heparin-and plateletbinding sites exist within the A3 domain (16 -18) and a binding site for factor XIIa is located in the A4 domain (19).Factor XI can participate in the contact phase of blood coagulation in a reaction that requires the presence of anionic surfaces for optimal activation in vitro by factor XIIa (6,[20][21][22]. However, deficiencies in factor XII, prekallikrein, and HK are not associated with hemostatic abnormalities, whereas a deficiency in factor XI produces abnormal bleeding complications (23-25). Therefore, it has been suggested that the physiologically relevant pathway for factor XI activation might constitute feedback activation either by thrombin or by factor XIa (7,8,15). All three proteases cleave each monomer of factor XI at the Arg 369 -Ile 370 bond generating the new amino-terminal sequence (IVGG) of the cataly...

Factor XI (FXI) 1 is a homodimeric plasma glycoprotein that circulates as a complex with its cofactor high molecular weight kininogen (HK) (1, 2) and is proteolytically activated on negatively charged surfaces by FXIIa to give rise to FXIa (3-10). The mechanism, involving interactions of FXII, prekallikrein (PK), and HK, by which contact activation is initiated and its significance in vivo have yet to be established, since individuals congenitally deficient in any one of these contact factors (FXII, HK, and PK) do not experience abnormal bleeding, suggesting that these proteins are not required for coagulation in vivo (11,12). In contrast, a deficiency of FXI can result in excessive bleeding after trauma or surgery (13,14). These observations suggest that FXI may be activated in vivo by a protease(s) other than FXIIa.The ability of thrombin, an enzyme generated late in the coagulation cascade, to activate FXI has been demonstrated (15,16). The site at which FXI is cleaved by thrombin is identical to that cleaved by FXIIa (16,17). Determination of the kinetic parameters of FXI activation by thrombin and FXIIa indicate that at a physiological concentration of FXI, in the presence of dextran sulfate, thrombin would be the more potent activator (16). Although FXI is readily activated by thrombin in a purified system with dextran sulfate present, the reaction may not proceed as readily in plasma (15,16,18), since although HK promotes the FXIIa-mediated reaction it inhibits thrombin-catalyzed activation of FXI (15,16,18). These observations raise the following two related questions. Is thrombin a physiological activator of FXI in plasma? What is the mechanism by which HK can inhibit thrombin-catalyzed FXI activation?The present study was undertaken to determine the sequence of amino acids in FXI that mediate its interaction with thrombin. Clarification of the mechanism of interaction of these two proteins might also help to elucidate the physiological importance of thrombin-catalyzed FXI activation. Four tandem repeat sequences (designated A1, A2, A3, and A4 or Apple domains) are present in the heavy chain of FXI (7). We have previously reported evidence for the presence of an HK binding site in the A1 domain (19,20), a binding site for FXIIa in the A4 domain (21), a substrate binding site for FIX in the A2 domain (22), and recently, a specific binding site for platelets in the A3 domain (23). Evidence for a binding site in the A1 domain of FXI that is important for interaction with thrombin is reported in the present study. were prepared in Escherichia coli using the QIAexpress pQE-9 expression vector (Qiagen Inc., Chatsworth, CA). PK was purified as described (25). FXI was assayed by minor modifications (26) of the kaolin-activated partial thromboplastin time (27). Human ␣-thrombin (2,800 NIH units/mg) was purchased from Enzyme Research Laboratories (South Bend, IN). All purified proteins appeared homogeneous by SDS-polyacrylamide gel electrophoresis. MATERIALS AND METHODS Purification of Proteins-FXIPeptide Synth...

To localize the platelet binding site on factor XI, rationally designed, conformationally constrained synthetic peptides were used to compete with [(125)I]factor XI binding to activated platelets. The major platelet binding energy resided within the sequence of amino acids T(249)-F(260). Homology scanning, using prekallikrein amino acid substitutions within the synthetic peptide T(249)-F(260), identified a major role for R(250) in platelet binding. Inhibition of [(125)I]factor XI binding to activated platelets by the recombinant Apple 3 domain of factor XI and inhibition by unlabeled factor XI were identical, whereas the recombinant Apple 3 domain of prekallikrein had little effect. A "gain-of-function" chimera in which the C-terminal amino acid sequence of the Apple 3 domain of prekallikrein was replaced with that of factor XI was as effective as the recombinant Apple 3 domain of factor XI and unlabeled factor XI in inhibiting [(125)I]factor XI binding to activated platelets. Alanine scanning mutagenic analysis of the recombinant Apple 3 domain of factor XI indicated that amino acids R(250), K(255), F(260), and Q(263) (but not K(252) or K(253)) are important for platelet binding. Thus, the binding energy mediating the interaction of factor XI with platelets is contained within the C-terminal amino acid sequence of the Apple 3 domain (T(249)-V(271)) and is mediated in part by amino acid residues R(250), K(255), F(260), and Q(263).

Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na ؉ -binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na ؉ -binding site (E229A and R233A) with <10% of the wildtype activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na ؉ -binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ib␣ (GPIb␣). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb␣ molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI␣ molecule, via ABE-I on its anterior surface. GPIb␣ plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.