Peter N. Walsh The Sol Sherry Thrombosis Research Center, Departments of Medicine and Biochemistry, Temple University School of Medicine, Philadelphia, PA, USA Correspondence to: Peter N. Walsh, M.D., Ph.D., Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA — Tel.: (215) 707-4375; Fax: (215) 707-3005; E-mail: pnw@astro.ocis.temple.edu *This study was supported by research grants from the National Institute of Health (HL46213, HL56914, and HL64943) Key words Factor XI, factor IX, protease nexin II, factor XII, high Mr kininogen Summary To account for the variable hemostatic defect in patients with factor XI (FXI) deficiency, with normal hemostasis in contact factor deficiencies, a coagulation paradigm is presented whereby trace quantities of thrombin, generated transiently by exposure of tissue factor at sites of vascular injury, activates FXI bound to the platelet surface in the presence of prothrombin or high Mr kininogen (HK). Tissue factor pathway inhibitor (TFPI) limits the flux of thrombin generated by the tissue factor pathway, and protease nexin II (PNII), released from activated platelets, inhibits solution phase FXIa and localizes FIX activation to the platelet surface where FXIa is protected from inactivation by PNII. Either prothrombin or HK binds to the Apple 1 (A1) domain of FXI, thereby exposing a platelet-binding site in the FXI A3 domain. Dimeric FXI binds to activated platelets directly through the A3 domain of one monomer. After proteolytic activation of platelet- bound FXI by thrombin (or FXIIa), a substrate binding site for FIX is exposed in the opposite monomer that promotes FIX activation on the platelet surface resulting in the local explosive generation of thrombin and the formation of hemostatic thrombi at sites of vascular injury. Initiation and Consolidation Phases of Blood Coagulation Classically, the sequence of coagulation reactions referred to as the coagulation cascade (1) or waterfall (2) has been understood as two alternative or convergent pathways activated either by the contact system proteases (3), factor XII (FXII), prekallikrein (PK) and high Mr kininogen (HK), or alternatively by the activation of FVII and FX when tissue factor is exposed at sites of vascular injury. Following the demonstration that a variety of cell membranes, including those exposed upon vascular injury (4), and those exposed when platelets are activated (5, 6), were required for many blood coagulation reactions, coagulation was conceived as the assembly of various protease-cofactor complexes assembled on biological membranes, particularly platelets where thrombin was generated in sufficient concentrations to convert fibrinogen to fibrin (6). This scheme required that FXI is activated by the equential activation of the contact proteins, FXII, PK, and HK. A major problem arising from this view of the classical sequence of coagulation reactions is that patients with FXI deficiency frequently present with abnormal bleeding complications after trauma or surgical operations (7-14) whereas patients with deficiencies of the contact proteins (FXII, PK and HK) do not experience abnormal hemostasis (3). Therefore, the question arose: "By what mechanisms and enzymes is FXI activated in vivo?" This question was addressed by both Naito and Fujikawa (15) and by Gailani and Broze (16) in 1991 who simultaneously showed that FXI could be activated not only by FXIIa but also by thrombin, albeit at very high concentrations and over protracted periods of incubation. However, in the presence of dextran sulfate the rate of FXI activation was significantly increased, and the concentra- tion of thrombin required to activate FXI was significantly reduced (15, 16). This observation gave rise to the important revised hypothesis for the initiation and propagation of coagulation initially by tissue factor exposed at sites of vascular injury resulting in the generation of small quantities of FXa which are required to facilitate the inactivation of FVIIa and FXa by TFPI (17-23). Thereby very small quantities of thrombin are generated resulting in the activation of FXI, the conver- sion of FIX to FIXa, and the subsequent activation of FX leading to the generation of sufficient amounts of thrombin to effect normal hemo- stasis (15, 16). Potential problems with this hypothesis at the time it was originally put forward included the lack of evidence that any physio- logical cell surface could substitute for dextran sulfate in vivo and the observation that HK, which forms a complex with FXI in plasma, could very effectively shut down the activation of FXI by thrombin (15, 16). This paper focuses on the mechanisms by which platelets and FXI participate in the consolidation (intrinsic) phase of blood coagulation leading to the generation of sufficient quantities of thrombin to effect normal hemostasis (24-36). Also considered here is the possible role of platelet FXI (9, 33, 37-39), an alternatively spliced product of the FXI gene expressed in a tissue specific manner in megakaryocytes and platelets, in blood coagulation and hemostasis. Finally, the expression of FXIa activity on the surface of activated platelets as well as its regulation by the Kunitz inhibitor, protease nexin II (PNII), are discussed (40-43). Plasma Factor XI Gene and Protein Structure The primary sequence and domain structure of plasma FXI are shown in schematic form in Figure 1. Plasma coagulation FXI circulates in plasma at a concentration of ~30 nM as a disulfide linked homodimer (Mr ~143,000) containing about 5% carbohydrate (44-48). The protein is encoded by a gene located on chromosome 4 (4q35), a 23 kb gene containing 15 exons and 14 introns encoding for a mRNA consisting of 2,097 nucleotides and a protein of 607 amino acids (49-51). Exons III-X encode four tandem repeat sequences (Apple domains) homologous to similar domains found in human plasma PK (58% identity) as well as in the N-terminal domains of plasminogen, hepatocyte growth factor and numerous nematode proteins, referred to as the PAN module (52), and in a microneme protein from Eimeria tenella (53). Exons XI-XV encode the typical trypsin-like catalytic domain which is activated by proteolytic cleavage of the zymogen at an internal Arg 369-Ile 370 bond to yield a heavy chain containing four Apple domains (369 amino acids) and the light chain or catalytic domain (238 amino acids). As shown in Fig. 1, each Apple domain contains six or seven cysteine residues that are highly conserved with similar disulfide-bonding patterns in FXI, PK and other PAN modular proteins (49, 50, 52). Amino acid sequence and domain structure of human plasma FXI. The single letter amino acid code shows the primary sequence of the protein containing a signal sequence (-18 to -1) which is removed during biosynthesis by a signal peptidase. The exons are denoted by Roman numeral designation from exon II to exon XV with exon II representing the propeptide and exon I (not shown) representing the 5’ untranslated region. The four Apple domains are shown (each con- sisting of 90-91 amino acids) with the subdomains implicated in various protein-protein and protein-cell interactions designated. The three members of the catalytic triad (H413, D462 and S557) are circled in bold. The four N-linked carbohydrate chains (N72, N107, N432, and N473) are shown by solid diamonds. The locations of the 14 introns (A-N) are shown by solid arrows. The asterisk adjacent to C321 indicates a single disulfide bond linking the A4 domains to form a homodimer. See text for details Molecular and Cellular Interactions Factor XI interactions with high molecular weight kininogen and platelets. Plasma FXI circulates in plasma in a noncovalent complex with HK (54) that promotes the binding of FXI to negatively charged surfaces (55) and its activation by its cognate proteases, FXIIa, FXIa, and thrombin, each of which can cleave a FXI monomer at the scissile bond Arg 369-Ile 370 (15, 16, 44). The HK binding site within FXI has been mapped to residues Phe 56-Ser 86 within the Apple 1 (A1) domain and residues Val 64 and Ile 77 have been implicated in this interaction (56-58). The molecular domains mediating ligand interactions with the A1 domain are summarized in Fig. 1. Complex formation with HK in the presence of Zn2+ ions has been shown to promote the binding of FXI to activated platelets (31, 33, 34, 36, 39, 59-63). The interaction of FXI with the surface of activated platelets has been shown to be mediated via residues Ser 248-Val 271 within the A3 domain of FXI (Fig. 1). Residues Ser 248, Arg 250, Lys 255, Phe 260 and Gln 263 have also been implicated in this interaction (27, 64-66). The A3 domain of FXI also contains a heparin binding site within residues Thr 249-Phe 260 and residues Lys 252 and Lys 253 have been implicated in the binding to platelets (67, 68). Although FXI and HK circulate in a noncovalent complex in plasma, and HK has been shown to bind to the surface of activated platelets, the interaction of FXI with the platelet surface apparently does not require binding of the HK-FXI complex. Instead, it appears that the FXI dimer binds directly to a high-affinity, specific site on activated platelets (n ~1,500 sites/platelet; Kd ~10 nM), since a 31 amino acid peptide from HK that binds directly to the A1 domain of FXI itself facilitates binding of FXI to activated platelets. The isolated recombinant A3 domain of FXI binds to the same number of sites on activated platelets and with the same affinity as the FXI dimer (65, 66). Factor XI interactions with prothrombin and platelets. Interestingly, although HK forms a high-affinity, reversible (Kd ~10-8 M) complex with FXI in plasma and promotes its binding to activated platelets in the presence of Zn2+ ions, prothrombin has also been shown to interact with FXI (Kd ~2.5 3 10-7 M) and to promote the binding of FXI to activated platelets in the presence of 2 mM Ca2+ (28, 30, 65, 69). This interaction of prothrombin with FXI is mediated via binding of the Kringle II domain of prothrombin (see Fig. 1) with residues Ala 45-Ser 86 within the A1 domain of FXI (69). Thus, prothrombin (1.5 mM) and Ca2+ (2 mM) can substitute for HK (45 nM) and Zn2+ (25 mM) in promoting FXI binding to activated platelets (28, 65). Factor XI activation by thrombin and factor XIIa on activated platelets. The functional consequences of the binding of FXI to activated platelets are a large enhancement of the rate of FXI activation by either FXIIa or by thrombin (28-30). A direct comparison of these two activators of FXI demonstrates that whereas the preferred activator of FXI in the presence of dextran sulfate is FXIIa, in the presence of activated platelets, thrombin is the preferred activator of FXI (30). Thus, the initial studies of thrombin as an activator of FXI demonstrated that FXI was very slowly activated by large concentrations of thrombin in the absence of dextran sulfate whereas in the presence of dextran sulfate, the concentrations of thrombin required for FXI activation were much lower (~1 nM) and the rates of activation were increased ~2,000-fold (15, 16). However, the presence of HK at plasma concentrations (~640 nM) very effectively shut down FXI activation by thrombin in the presence of dextran sulfate (15, 16, 70, 71). In contrast, in the presence of platelets, HK promotes FXI binding to activated platelets and greatly promotes FXI activation by thrombin (28-30) and by FXIIa (28, 30, 31, 33, 34, 36, 39, 59-63). FXIIa has been shown to interact with a subdomain (Ala 317-Gly 350) within the A4 domain of FXI that is involved in the activation of FXI by FXIIa (72). In addition, a thrombin binding site has been identified within the A1 domain of FXI comprising a sequence of amino acids, Ala 45-Arg 70, and Asp 51 and Glu 66 have been implicated in the binding of active site inhibited thrombin to the A1 domain of FXI (73). We have concluded from these observations that prothrombin and calcium ions can substitute for HK and zinc ions in promoting the binding of FXI to activated platelets and that thrombin is the preferred activator of FXI on the platelet sur- face (30), thereby obviating the requirement for contact factors (FXII, PK and HK). This may explain the requirement for FXI in normal hemostasis since FXI deficiency is associated with hemostatic abnormalities whereas deficiencies of the contact factors (FXII, PK and HK) are not associated with abnormal bleeding (3, 7-14). Expression of factor XIa activity. The active enzyme, FXIa, has been shown to bind to high-affinity, saturable (Kd ~800 pM; n ~500 sites/ platelet), specific sites on activated platelets (31, 33, 34, 36, 74, 75). When bound to the platelet surface, FXIa can activate FIX albeit at rates similar to those observed in solution (75). Studies with mono- clonal antibodies have demonstrated the presence of a macromolecular substrate binding site for FIX within the heavy chain region of the enzyme that is essential for the calcium-dependent activation of FIX (76-79). Both a subdomain (Ala 134-Leu 172) in the A2 domain (76) and two subdomains (Ile 184-Val 192 and Ser 259-Ser 265) within the A3 domain (80, 81) have been proposed as comprising this substrate (FIX)-binding site. Dimerization of factor XI (XIa). One of the more interesting characteristics of FXI is its homodimeric structure, which raises questions of functional significance. Meijers and coworkers demonstrated that at least a portion of the molecular information required for mediating dimer formation between the two identical subunits resides within the A4 domain of FXI since chimeric tissue plasminogen activator (tPA) molecules, with the A4 domain of FXI substituted for the finger and growth factor domains, exist as dimers by gel filtration (82, 83). Cys 321 within the A4 domain has been shown to mediate covalent homodimer formation (50, 84), but when Cys 321 is replaced by serine, FXI forms a noncovalent dimer suggesting that the A4 domains mediate noncovalent interactions resulting in dimer formation which is stabilized by a covalent linkage between cysteine residues in each monomer at position 321 (82, 83, 85). Recently we have shown that the recombinant A4 domain of human plasma FXI also forms covalent homo- dimers and that when Cys 321 is replaced by a serine, a slowly equilibrating, reversible monomer-dimer equilibrium is established characterized by a Kd value of 229 ± 26 nM with a calculated DG value of 9.1 kcal/mol (85). Recently we have examined the possible functional significance of homodimer formation by preparing a monomeric ver- sion of FXI with the A4 domain replaced by that of PK that exists in plasma as a monomer (86). Both monomeric and dimeric FXIa molecules activated FIX with kinetic parameters similar to those of normal wild-type or plasma-derived FXIa, and both monomeric and dimeric molecules were shown to have similar coagulant activity in the presence of phospholipid membranes (86). Although both monomeric and dimeric FXIa molecules demonstrated similar binding affinities for activated platelets, the monomeric protein was unable to activate FIX in the presence of activated platelets suggesting that monomeric FXIa is unable to interact simultaneously with activated platelets and with its normal macromolecular substrate, FIX (86). The results suggest a model in which FXIa binds to the platelet surface utilizing one polypeptide chain of the dimer thereby presenting the other monomer as a substrate binding site for FIX (86). Regulation of factor XIa by protease nexin II. The observations summarized above strongly suggest that the physiological site of activation of FXI and formation of the enzyme, FXIa, is the activated platelet membrane where specific high-affinity (Kd ~800 pM) saturable receptors (~500 sites/platelet) for FXIa are generated in the presence of HK (31, 33, 34, 36, 74, 75). Since both FXIa (74) and FIX (25) can bind to high-affinity saturable receptors on activated platelets, and FIX activation by FXIa can occur on the platelet surface (75), it is likely that FIXa generation serves to localize FIXa-catalyzed FX activation to the platelet surface (24-26, 32) which also promotes prothrombin activa- tion by FXa (87). In addition to forming membrane associated complexes leading to the local explosive generation of thrombin on the platelet surface, FXIa is also subject to regulation by a variety of plasma and platelet protease inhibitors whose functional activity appears to depend on whether FXIa is bound to the platelet surface or whether it is free in solution. Thus, a number of serine protease inhibitors including a-1-protease inhibitor, antithrombin III, C1 inhibitor, a-2-antiplasmin, plasminogen activator inhibitor 1 and protein C inhibitor have all been shown to inactivate FXIa in the plasma compartment (45, 46, 88-99). However, within the environment of activated platelets, it seems likely that the most physiologically relevant inhibitor of FXIa is PNII, a truncated form of the transmembrane Alzheimer’s amyloid b-protein precursor, which contains a Kunitz-type serine protease inhibitor domain (40-43, 100-104). Thus, PNII is found in very low concentra- tion in plasma but is secreted from platelet a-granules (1-1.5 nM PNII released per 108 platelets) such that at physiological platelet concentration, the plasma concentration of PNII may be brought to 3-5 nM (40, 104). PNII is a potent inhibitor of FXIa with a Ki of 300-500 pM that is significantly enhanced (Ki ~30 pM) in the presence of heparin (42, 43, 100, 102, 104). We have shown that FXIa bound to the platelet surface in the presence of HK and Zn2+ ions is protected from inactivation by both PNII (42) and also by a-1-protease inhibitor (99) giving rise to the suggestion that FXIa activity generated on the platelet surface is localized to the hemostatic thrombus whereas the site of regulation of FXIa by PNII and other protease inhibitors occurs in free solution (42, 99). In addition, heparin enhances the inactivation of FXIa by PNII by a template mechanism involving the colocalization of both PNII and the catalytic domain of FXIa on a single strand of full-length heparin (43). It is also possible that endothelial cells, which contain heparan sulfate glycosaminoglycans, might promote the assembly of FXIa/PNII complexes thereby potentiating the inhibition of FXIa on the endothelium. The role of factor XI in fibrinolysis. The foregoing discussion emphasizes the clinical observation that patients with FXI deficiency are prone to bleeding complications, whereas patients with contact factor deficiencies (FXII, PK and HK) are not subject to any increased risk of bleeding even after major trauma (3, 7-14). This clinical ob- servation supports the conclusion that the major pathway for FXI activation on the platelet surface is thrombin generation via the FVII-tissue factor pathway that is rapidly down-regulated by TFPI (15, 16). It has also been observed that patients with FXI deficiency, most common among Ashkenazi Jews, are especially prone to bleeding from tissues with high local fibrinolytic activity including the urinary tract, the nose, the oral cavity and the tonsils (7-12, 105-108). This observation has given rise to the suggestion that FXI might play a role in the down-regulation of fibrinolysis (109). This postulated antifibrinolytic activity of FXI is thought to occur as a consequence of a FXI-dependent burst in thrombin generation resulting in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) or procarboxypeptidase B which inhibits the activation of plasminogen by removing carboxy-terminal lysines from fibrin that are essential for plasminogen binding and activation (109-114). The evidence supporting a role for FXI in the down-regulation of fibrinolysis through the activation of TAFI and through other thrombin-mediated mechanisms is summarized elsewhere (115). Platelet Factor XI A particularly intriguing problem relating to the physiology of FXI is the fact that the bleeding tendency in FXI deficient patients is variable with ~50% of patients exhibiting excessive post-traumatic or post-surgical bleeding whereas the remainder appear to be hemostatically normal (7-14, 105-108). A possible explanation for this variable phenotype of FXI deficiency is that a second form of FXI found in the platelets of normal individuals might compensate for the absence of plasma FXI, thus explaining the absence of bleeding complications in certain individuals with plasma FXI deficiency (9, 11, 38, 116, 117). Platelet FXI has been detected as a FXI-like coagulant activity and as the presence of FXI antigen in well washed platelet suspensions, accounting for ~0.5% of the FXI activity in normal plasma, from which it can be calculated that there are ~300 molecules of platelet FXI per platelet (116, 118-120). Platelet FXI activity is enriched in the plasma membrane fraction (38). Platelet FXI appears to differ structurally from plasma FXI since three separate groups (116, 118, 120) have demonstrated by SDS-polyacrylamide gel electrophoresis that platelet FXI has an apparent Mr of ~220,000 (55,000 after reduction) whereas plasma FXI has a Mr of 160,000 (80,000 reduced). Recent observations from our laboratory have demonstrated using reverse transcriptase-poly- merase chain reaction (RT-PCR) of platelet mRNA, Northern blot analysis and screening of a cDNA library from a megakaryocytic cell line (CHRF-288) that platelet FXI mRNA is identical to mRNA for plasma FXI with the exception of the absence of exon V (37). These results suggest that platelet FXI is the product of an alternatively spliced FXI mRNA or possibly the product of a second FXI gene lacking exon V and expressed in a tissue-specific manner in megakaryocytes and platelets (37). In contrast, Martincic et al. (121) while confirming the presence of FXI mRNA amplified by RT-PCR from platelets and megakaryocytes, reported the recovery of only a full-length FXI in mRNA that is identical to mRNA from liver. The presence of an identical gene product in both liver and megakaryocytes would not explain the apparent difference in Mr between platelet FXI and plasma FXI (116, 118, 120), nor would it account for the presence of platelet FXI in a subset of patients with plasma FXI deficiency (9, 38, 116, 120). In an attempt to resolve this apparent controversy, we have examined the platelets of four unrelated patients with severe deficiency of plasma FXI without bleeding complications even after trauma or surgery (9). Utilizing flow cytometry and a functional assay for platelet FXI, we have detected normal constitutive and activation-dependent expression of platelet FXI suggesting that the tissue-specific expression of functional platelet FXI is independent of plasma FXI expression (9). A possible explanation for normal expression of platelet FXI in the absence of plasma FXI arises from a characterization of the genetic mutations leading to FXI deficiency. Thus, it has been shown that the majority of patients with plasma FXI deficiency among individuals of Ashkenazi Jewish ancestry have either a termination codon in exon V (Type II FXI deficiency) or alternatively, a missense mutation in exon IX (Type III FXI deficiency), resulting in a Phe 283 Leu substitution in the A4 domain of FXI (7, 82, 83, 122-129). Genotypic analysis of three Ashkenazi Jewish patients with severe plasma FXI deficiency, normal hemostasis, and normal levels of platelet FXI demonstrated the presence of a Type II mutation in one or both alleles of all three patients leading to a stop codon in exon V (130). Thus, it is possible that the presence of a premature termination codon in exon V resulting in the virtual ab- sence of plasma FXI is consistent with normal production of platelet FXI which can compensate for the absence of plasma FXI and result in normal hemostasis. It should be emphasized that there may be other explanations for the variable phenotype observed in patients with plasma FXI deficiency. The physiological role of platelet FXI remains undefined including its mechanism of activation, the expression of its enzymatic activity and its role in blood coagulation in relation to plasma FXI. The Role of Factor XI in Hemostasis The fact that patients with FXI deficiency have a variable hemo- static defect emphasizes the importance of FXI in promoting blood coagulation and maintaining normal hemostasis in contrast to the proteins of the contact phase of blood coagulation (FXII, PK and HK), whose deficiencies are not associated with abnormal hemostasis and bleeding (3, 7-14). The observations summarized in this paper support the view, summarized in schematic form in Fig. 2, that the initiation of hemostasis most likely normally occurs through the exposure of tissue factor at sites of vascular injury resulting in the generation of small quantities of FXa that allow TFPI to function and limit the flux of thrombin generated via the tissue factor pathway (15, 16, 30). Fig. 2   A model of the sequence of reactions occurring during the initiation and the consolidation of blood coagulation. See text for explanation. The crescentic forms represent the cell membranes that localize various coagulation reactions, with the tissue factor/factor VIIa complex assembled on tissue factor bearing cells (gray filled membrane) and the remainder of the complexes assembled on the activated platelet membrane (unfilled crescent). The Roman numerals represent coagulation proteins in the zymogen or cofactor form with the "a" representing the active enzyme or cofactor. The circles represent zymogens: the circles with segmental excisions represent enzymes; the ellipses represent cofactors; and, the rectangles represent Kunitz-type inhibitors with the solid block representing the block imposed by that inhibitor. The designation pXI represents platelet factor XI. The arrows represent conversions from zymogens to enzymes. Other abbreviations include: HK, high molecular weight kininogen; TFPI, tissue factor pathway inhibitor; PN2, protease nexin II; II, prothrombin; TF, tissue factor However, the concentration of thrombin thus formed appears to be sufficient to result in the activation of platelets to expose receptors for FXI, which can be activated on the platelet surface by very small quantities of thrombin (28, 30). The fact that activated platelets can also promote the proteolytic activation of FXI by FXIIa suggests the possibility that in normal individuals, some FXIa generation may occur on the platelet surface via activation of the contact proteins when platelets are activated by ADP, collagen or other activators (33, 34, 36). However, the fact that prothrombin can substitute for HK and that thrombin is the pre- ferred activator of FXI on the platelet surface also suggests the possibility that under normal physiological conditions, FXI-dependent coagulation occurs via contact factor independent pathways (28, 30). The role of platelet FXI in these molecular interactions is currently un- known (9, 33, 37-39). FXIa generated on the platelet surface appears to be protected from inactivation by PNII, which is likely to be the major regulator of FXIa activity in solution (42). FXIa generation on the platelet surface promotes FIX activation leading to FIXa generation (24-26, 32). 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