Prekallikrein: the catalytic crossroads of inflammation and thrombosis

Abstract

Purpose of review

The contact activation pathway, historically considered dispensable for hemostasis, has re-emerged as a critical mediator of thromboinflammation. Plasma prekallikrein, through its activation to kallikrein, links coagulation, inflammation, and vascular biology. This review is timely given increasing interest in targeting the contact system to develop safer antithrombotic therapies that minimize bleeding risk.

Recent findings

Emerging preclinical and translational studies demonstrate that prekallikrein contributes to thrombosis, inflammation, and endothelial dysfunction, despite minimal involvement in baseline hemostasis. Animal models reveal context-dependent roles of prekallikrein in arterial and venous thrombosis, while human data confirm that prekallikrein deficiency is not associated with bleeding. Clinical success of prekallikrein inhibition in hereditary angioedema validates the therapeutic relevance of the kallikrein–kinin system. Early-phase studies and related work targeting contact pathway components, particularly factor XI, further support this pathway as a viable antithrombotic target.

Summary

Collectively, current evidence positions prekallikrein as a promising therapeutic target that may allow selective inhibition of thromboinflammatory processes without impairing hemostasis. Further clinical investigation is needed to define the efficacy and safety of prekallikrein-directed therapies in thrombosis and other inflammatory conditions.

INTRODUCTION

Plasma prekallikrein is a component of the contact activation pathway, a branch of the coagulation cascade that serves as a molecular link between coagulation and inflammation [1]. The contact pathway is initiated when blood encounters negatively charged or artificial surfaces, triggering a proteolytic cascade involving factor XII (FXII), factor XI (FXI), high-molecular-weight kininogen, and prekallikrein. Together, these proteins form a tightly regulated system that can promote both thrombin generation and inflammatory signaling, particularly through the activation of the kallikrein–kinin system and the release of bradykinin, a potent vasoactive peptide [2].

Although prekallikrein deficiency is detectable by a prolonged activated partial thromboplastin time (aPTT), individuals with congenital prekallikrein deficiency do not exhibit spontaneous bleeding [3]. Similarly, deficiencies in FXI and FXII are not associated with an unprovoked bleeding diathesis [4^■]. These clinical observations underscore that the contact pathway is largely dispensable for physiologic hemostasis; yet, the activation of prekallikrein has been increasingly implicated in pathologic thrombosis and inflammation [5]. For instance, the clinical relevance of prekallikrein has been clearly demonstrated in hereditary angioedema (HAE), a condition driven by excessive bradykinin production [6]. Targeted inhibition of prekallikrein has proven to be both effective and well tolerated in this setting, implicating prekallikrein as a druggable target [7,8^■■]. This success has sparked broader interest in understanding the role of prekallikrein and the contact system more generally in thromboinflammatory disease.

Despite recent preclinical studies suggesting that activation of prekallikrein may actively promote thrombosis in animal models, whether limiting prekallikrein activation prevents thrombosis in humans is unstudied [9]. Motivation and hope for contact pathway inhibitors as safe antithrombotic agents is provided by the successful reduction of thrombosis observed in several clinical trials of FXI inhibitors [10^■,11,12]. In fact, these findings have given rise to a new therapeutic paradigm: targeting components or enzymatic steps of the contact pathway to selectively inhibit thrombosis while minimizing the risk of bleeding. Anticoagulation remains the cornerstone of thrombosis prevention and treatment, with over 6 million patients in the United States alone receiving anticoagulants annually [13]. However, current therapies carry a substantial bleeding risk, highlighting the urgent need for safer, more targeted approaches [14,15]. The contact activation pathway inclusive of FXI, FXII, prekallikrein, and high-molecular-weight kininogen has thus emerged as a promising focus for safer next-generation antithrombotic therapies.

In this review, we summarize the current body of knowledge on the biology of prekallikrein, including its mechanistic roles in coagulation and inflammation. We highlight preclinical data supporting prekallikrein’s contribution to thrombosis and examine early clinical findings related to prekallikrein inhibition. Collectively, these findings suggest that prekallikrein may represent a novel, well tolerated, and effective therapeutic target for thrombotic and thromboinflammatory disease.

THE BIOLOGY OF PREKALLIKREIN

Prekallikrein is a single-chain glycoprotein that circulates as the inactive zymogen of the active serine protease plasma kallikrein [16]. As a member of the kallikrein–kinin system, the appearance of prekallikrein immediately preceded the evolutionary step of migration to land. Prekallikrein first arose in lobe-finned fish such as the lungfish and is rather ubiquitously expressed among terrestrial vertebrates [17]. The loss of prekallikrein along with FXII protein expression in cetaceans (the order of aquatic mammals which includes dolphins and whales) due to the accumulation of gene-inactivating mutations in the KLKB1 as well as the F12 genes, respectively, may have conferred a survival advantage by removing the kallikrein–kinin system during the mammalian return to the sea [18]. Alternatively, KLKB1 and F12 may have suffered the evolutionary pruning of genes that arose through duplication, as the gene for FXII evolved from the duplication of the hepatocyte growth activator (HGFA) gene while the duplication of the KLKB1 gene gave rise to FXI [19].

The structure of prekallikrein is homologous to the protein structure of coagulation factor (F) XI, both consisting of four apple domains and one serine protease domain (Fig. 1) [19]. The majority of prekallikrein and almost all FXI circulate as stable complexes with high-molecular-weight kininogen. Initial molecular modeling studies predicted that high-molecular-weight kininogen bound the A1 and A2 domains of prekallikrein [20]. Structural biology studies using a 31-amino acid peptide spanning the Ser565-Lys595 residues of the high-molecular-weight kininogen D6 domain confirmed that binding of high-molecular-weight kininogen to the A1 and A2 domain of prekallikrein is mediated by the high-molecular-weight kininogen sequences WIPDIQ (Trp569-Gln574) and FNPISDFPDT (Phe582-Thr591), respectively, with a flexible intervening sequence that potentiates a bent double conformation [21]. Notably, earlier functional studies demonstrated that the prekallikrein A2 domain plays a critical role in high-molecular-weight kininogen binding [22].

FIGURE 1.

Prekallikrein binding partners. Schematic of plasma prekallikrein illustrating binding partners across structural domains. The N-terminal apple domains (A1–A4) mediate protein–protein interactions, with high-molecular-weight kininogen (HMWK) binding primarily to A1 and A2 and facilitating surface localization. Activated factor XII (FXIIa) interacts with the A4 domain to enable reciprocal activation within the contact pathway. Additional reported prekallikrein-binding partners include C1 esterase inhibitor (C1-INH), urokinase-type plasminogen activator (uPA), heat shock protein 90 (HSP90), prolylcarboxypeptidase (PRCP), and complement component C3, which interact predominantly with the catalytic or C-terminal regions. NH₂ and COOH denote the amino-termini and carboxyl-termini. A1–A4, apple domains 1–4; C1-INH, C1 esterase inhibitor; C3, complement component 3; FXIIa, activated factor XII; HMWK, high-molecular-weight kininogen; HSP90, heat shock protein 90; PK, prekallikrein; PRCP, prolylcarboxypeptidase; uPA, urokinase-type plasminogen activator.

As a core member of the contact system, prekallikrein is activated following the binding and activation of FXII by highly charged surfaces typically foreign to the blood microenvironment. The activation of prekallikrein by activated FXII (FXIIa) to form the enzyme kallikrein converts a faithful servant to a bad master. Kallikrein reciprocates this favor by activating FXII [23]. Platelet-derived polyphosphate serves as a catalyst to dramatically accelerate the kinetics of this vicious feedback loop of FXIIa and kallikrein generation [24]. The ability of kallikrein to cleave the central complement component C3 coupled with the ability of FXIa to neutralize complement factor H provides a mechanism by which the contact activation system synergizes with the amplification loop of the alternative pathway of complement [25]. More potently, kallikrein cleaves its once docile binding partner, high-molecular-weight kininogen, to generate the nonapeptide and potent vasodilator bradykinin [5]. As kallikrein activity is balanced by inactivation by the serpins antithrombin and C1 inhibitor (C1-Inh), the latter of which also regulates the classical complementary pathway. Deficiency or dysfunction of C1-Inh permits excessive generation of bradykinin associated with the increased blood vessel permeability and swelling characteristic of HAE [5]. Taken together, the molecular binding partners of prekallikrein and enzymatic activity of kallikrein suggest a key role for prekallikrein in initiating and amplifying the coagulation, immune, and inflammatory pathways.

THE FUNCTION OF PREKALLIKREIN: LESSONS FROM IN-VITRO MODELS

The creation of antibodies specifically targeting the active forms of contact factors, kallikrein and FXIIa, provided the necessary tools to delineate the function of prekallikrein in thrombin generation and fibrin formation [9]. Initial studies in plasma supplemented with DNA showed a marked (5–10-fold) increase in reciprocal activation of prekallikrein and FXII. The inhibitory antibodies IgG 559C-X181-D06 (D06, FXIIa inhibitor) and 559A-M202-H03 (H03, α-kallikrein inhibitor) both reduced DNA-induced prekallikrein and FXII activation [8^■■,9,26]. The potency of preventing thrombin generation by either silica or collagen was greater for D06 as compared to H03, suggesting that FXII serves as the main character in contact activation in a closed system, with prekallikrein playing the role of supporting cast [9]. Using truncated proteases, prekallikrein/FXI chimeric proteins, and antibodies to specific prekallikrein apple domains, we recently studied the function of the specific apple domains of prekallikrein in the absence or presence of a surface (the polyanion polyphosphate) [27,28]. We found that the PKA3 domain mediates the interactions with FXII and FXIIa, while the PKA1 and PKA2 domains are required for mediating interactions with high-molecular-weight kininogen. We found that prekallikrein antibodies directed against PKA1, PKA2, and PKA3 block generation of kalikrein, FXIIa, and bradykinin in plasma [28]. This link between bradykinin production and surface activation of prekallikrein may represent a therapeutic target to reduce tissue swelling and inflammation.

THE FUNCTION OF PREKALLIKREIN: LESSONS FROM IN-VIVO MODELS

Genetic knockout models of select members of the contact activation pathway allowed for the mechanistic investigation of the role of prekallikrein and FXII in arterial thrombus formation in mice (Table 1). In a ferric chloride carotid injury model, both FXII-deficient and homozygous prekallikrein-deficient mice were observed to be resistant to occlusion, with FXII deficiency offering protection from occlusion even at higher concentrations of FeCl₃. Heterozygous prekallikrein-deficient and kininogen-deficient mice showed only partial resistance as measured by thrombus weight and carotid blood flow rate [9]. Yet, in a Rose Bengal/laser injury model, while FXII deficiency markedly prolonged time to vessel occlusion, prekallikrein deficiency had no significant effect compared to wild type [9]. Likewise, genetic deletion of FXII but not prekallikrein provided a survival benefit in a lethal model of pulmonary embolism induced by either collagen/epinephrine or bacterial long-chain polyphosphate [29]. Overall, these initial in-vivo studies reinforced the paradigm of a supporting cast member role for prekallikrein in thrombus formation, overshadowed by the dominant performance of FXII.

Table 1.

Animal models

Study (year)	Population and model details	N (analyzed)	Intervention	Key results	Adverse events	Key limitations
Kokoye et al., 2016 [9]	FXII-deficient, PK-deficient, and HMWK-deficient mice	n = 10–15 per group	Anti-FXIIa (D06) and Anti-PK (H03) antibodies	FXII deficiency was more protective than PK deficiency; FXIIa inhibition was more potent in human flow models	No increase in provoked bleeding	Murine model translatability
Revenko et al., 2011 [30]	BALB/c or C57BL/6 mice	n = 4–10 per group	ASO-mediated knockdown of PK or fXII	Selective reduction of either factor inhibits thrombosis	No effect on hemostasis or bleeding risk	Dependence on ASO knockdown efficiency
Bird et al., 2012 [39]	PK-deficient mice, wild-type mice	n = 8 per group	Genetic ablation or PK ASO treatment	Complete protection from arterial occlusion; reduced venous thrombus weight	Modest increase in tail bleeding time	Rodent-specific hemostatic kinetics
Stavrou et al., 2015 [29]	PK-deficient mice	n = 7–12 per group	Genetic deletion; pharmacologic antagonists	Reduced thrombosis via increased Mas/prostacyclin and decreased vascular TF	No bleeding defect observed	Focus on murine-specific molecular pathways

ASO, antisense oligonucleotide; BALB/c and C57BL/6, inbred mouse strains; FXII, factor XII; fXII, factor XII; FXIIa, activated factor XII; HMWK, high-molecular-weight kininogen; KO, knockout; Mas, Mas receptor; PGI₂, prostacyclin; PK/PKK, prekallikrein; TF, tissue factor; WT, wild type.

Use of antisense oligonucleotides (ASOs) to selectively knockdown, but not eliminate, prekallikrein and FXII levels in mice revealed an interdependent relationship between these members of the contact pathway [30]. An upwards of a 95% reduction in the protein levels of either prekallikrein or FXII was achieved by selectively targeting each zymogen. At steady state, a reduction in prekallikrein levels caused an increase in circulating FXII levels, and correspondingly a decrease of activated FXII in complex with serpins including antithrombin. Conversely, reducing FXII levels caused an increase in circulating prekallikrein levels while reducing the degree of kallikrein in complex with serpins. In a mouse model of stenosis-induced inferior vena cava thrombosis, targeted reduction of prekallikrein produced a gradual dose-dependent antithrombotic response, requiring near-complete reduction in prekallikrein levels for full efficacy [30]. In this model, a highly potent and efficacious response was observed when FXII levels were reduced, with near-complete inhibition of thrombosis even when FXII levels were reduced by only 40%. The opposite was observed in models of arterial thrombosis, where a slight reduction in prekallikrein levels produced a potent antithrombotic effect, whereas near complete reduction of FXII was required to achieve the same level of antithrombotic efficacy. In both venous and arterial thrombosis models, clots were unstable and fragmented under flow [30]. Finally, in a tail vein bleeding assay, depletion of prekallikrein or FXII did not increase bleeding, in contrast to the doubling of tail bleeding times observed in prekallikrein-deficient mice or significant levels of bleeding observed when prothrombin mRNA was knocked down [30].

While the role of prekallikrein in hemostasis appeared minimum, prekallikrein-deficient mice were protected from lung edema-capillary leak in the models of lethal pulmonary embolism, as measured by Evans blue staining [29]. This suggests that perhaps prekallikrein may play a role in regulating endothelial barrier function, akin to the regulatory roles recently described for the prekallikrein homolog, FXI. Indeed, prekallikrein deficiency was associated with a decrease in bradykinin levels, reflective of the crosstalk between the kallikrein–kinin system and renin–angiotensin system. Mechanistically, prekallikrein deficiency reduced renal mRNA for bradykinin receptor 2, angiotensin II receptor type II, and angiotensin converting enzyme (ACE), resulting in an overexpression of the Mas receptor [29]. A role for the Mas receptor in mediating the prothrombotic role for prekallikrein was confirmed using a Mas antagonist, which reversed the antithrombotic phenotype observed for prekallikrein-deficient mice in a model of carotid artery occlusion [29]. Moreover, the increased expression of Mas receptor led to an increase in plasma prostaglandin levels, which was associated with an increase in the vasoprotective transcription factors Sirt1 and KLF4 and decreased tissue factor expression in the aorta [29]. These findings suggest that select functions of prekallikrein may represent pharmacological targets to reduce the prothrombotic phenotype of endothelial cells in the setting of inflammation.

HUMAN STUDIES EVALUATING PREKALLIKREIN DEFICIENCY: THE CLINICAL IMPACT OF FLETCHER FACTOR

Severe plasma prekallikrein deficiency, also known clinically as Fletcher factor deficiency, is an autosomal-recessive disorder that typically manifests as an isolated prolonged aPTT without significant bleeding symptoms [31]. This condition was first described in 1965 after a Kentucky family was found to have notably abnormal APTT results, despite the absence of bleeding symptoms [32]. The family appeared to have a hereditary deficiency in a yet unknown coagulation factor, which was subsequently named ‘Fletcher factor’ in their honor. Mutations in the KLKB1 gene were later found to be responsible for this deficiency [3].

Clinically, most individuals with severe prekallikrein deficiency are asymptomatic. Population-based studies, including data from the Genome Aggregation Database (gnomAD), estimate the global prevalence of severe prekallikrein deficiency at about 1 in 155 668, with a higher prevalence of 1 in 4725 among Africans [3]. Prekallikrein deficiency is usually discovered incidentally during routine coagulation testing obtained for other indications. Cardiovascular events are reported in about 15% of individuals with prekallikrein deficiency, with an annualized rate of 0.4%. Bleeding events are rare, with a prevalence of 4% and an annualized rate of 0.1%. While some rare bleeding events have been documented in prekallikrein-deficient patients, whether this is mechanistically linked to prekallikrein is uncertain, given the general lack of bleeding seen in animal models [6,8^■■,9,29].

HUMAN CLINICAL TRIALS TARGETING PREKALLIKREIN FOR NONTHROMBOTIC INDICATIONS

To date, four drugs directly targeting prekallikrein have been evaluated in human clinical trials of HAE: Sebetralstat [33^■■], Donidalorsen (formerly known as IONIS-PKK-LRx) [8^■■], Berotralstat (formerly known as BCX7353) [34], and Lanadelumab (DX-2930) [35] (Table 2). Sebetralstat is an oral prekallikrein inhibitor Food and Drug Administration (FDA)-approved for the treatment of angioedema attacks which binds directly to the active site of plasma kallikrein. Similarly, Donidalorsen, an antisense oligonucleotide targeting prekallikrein mRNA in the liver, which halts prekallikrein transcription, is under development as prophylaxis for angioedema [8^■■]. Berotralstat is a potent oral inhibitor of plasma kallikrein that helps to prevent HAE attacks and is currently FDA-approved [21]. Lastly, lanadelumab is a monoclonal antibody that binds to the catalytic domain of plasma kallikrein that is FDA -approved for the treatment of HAE [36^■].

Table 2.

Human Clinical Trials

Study (year)	Design/setting	Population and target	N (analyzed)	Intervention	Key results	Adverse events
Riedl et al., 2024 [33■■]	Phase 3, double-blind, three-way crossover trial	HAE patients (type I or II) age ≥12	136	Sebetralstat (300 mg, 600mg, orally) vs. placebo	24-h complete resolution: 42.5% (300 mg); 49.5% (600 mg); 27.4% (placebo)	No serious adverse events were reported
Riedl et al., 2024 [8■■]	Phase 3, RCT (double-blind)	Patients with type I or II HAE	90	Donidalorsen (80 mg every 4 or 8 weeks) vs. placebo	HAE attack rate reduction: 90% (Q4W), 82% (Q8W), placebo 16%	Erythema at the injection site, headache, and nasopharyngitis
Aygoren et al., 2018 [34]	Phase 2, RCT (double-blind)	Patients with type I or II HAE	75	Berotralstat (62.5, 125, 250, 350 mg oral daily) vs. placebo	~74% reduction in HAE attack rate at 125 mg dose	Primarily mild GI symptoms
Banerji et al., 2018 [36]	Phase 3, RCT (double-blind)	HAE patients (type I or II) age ≥12	125	Lanadelumab (300 mg s.c. every 2 weeks) vs. placebo	87% reduction in mean monthly HAE attack rate	Injection site pain, erythema, and headache

GI, gastrointestinal; HAE, hereditary angioedema; N, number analyzed; Q4W, every 4 weeks; Q8 W, every 8 weeks; RCT, randomized controlled trial; s.c., subcutaneous.

The Sebetralstat Phase 3 clinical trial (the KONFIDENT trial) was a double-blinded triple crossover study for the treatment of acute angioedema attacks, in which one group received 600mg (two pills) of Sebetralstat, while another group was given 300mg (one pill + one placebo pill), and the final group was given a basic placebo (two placebo pills) [33^■■]. The trials showed a significant improvement in median angioedema attack recovery times 1.61, 1.79, and 6.72h where the percentage of attacks that had been resolved within 24h were 49.5, 42.5, and 27.4% respectively [33^■■].

There have been several studies assessing the impact of Donidalorsen for the treatment of HAE. Following successful phase 1 and 2 studies, which included an endpoint demonstrating a dose-dependent increase in aPTT [6,38], Donidalorsen was trialed in a phase 3 study. The OASIS-HAE trial showed a reduction in angioedema in attacks by 90% with the use of Donidalorsen as compared to a 16% reduction in the placebo arm [8^■■].

Berotralstat was evaluated in the APeX-1 trial. This phase 2, randomized, double-blind study showed that individuals receiving 125mg or more of BCX7353 exhibited a significant decrease in angioedema attacks with the only major side effect being some minor gastrointestinal symptoms [34].

Lastly, Lanadelumab was FDA-approved following assessment in a phase 3 clinical trial. Lanadelumab at a dose of 300 or 400mg reduced cleavage of high-molecular-weight kininogen in plasma from patients with HAE with C1 inhibitor deficiency to levels approaching that from patients without the disorder. The 300 and 400-mg groups had 100 and 88% fewer attacks, respectively, than the placebo group. All patients in the 300-mg group and 82% (9 of 11) in the 400-mg group were attack-free, as compared with 27% (3 of 11) in the placebo group [35].

CONCLUSION

Although prekallikrein deficiency may be clinically silent, prekallikrein appears to contribute to various thromboinflammatory disorders through dual activation of the contact pathway and the kallikrein–kinin system. Animal studies using strategies to genetically knockout or knockdown prekallikrein or pharmacological target select regions of prekallikrein have largely highlighted a role for prekallikrein in thrombosis and inflammation largely in the absence of a role in hemostasis. Clinical studies confirm that prekallikrein is mostly dispensable for normal hemostasis, as individuals with prekallikrein deficiency generally do not exhibit a bleeding phenotype. Clinical trials utilizing small molecules, antisense oligonucleotides, and monoclonal antibodies to treat HAE have validated a role for prekallikrein in the inflammatory pathology underlying angioedema. It will be exciting to see whether targeting prekallikrein will be useful for reducing inflammation in patients requiring medical devices or with bacterial sepsis, both pathologies associated with surface activation of the contact pathway.

KEY POINTS.

• Structural mapping of plasma prekallikrein’s apple domains reveals distinct binding interfaces for FXIIa and high-molecular-weight kininogen, identifying mechanistic targets for selective contact pathway inhibition.

• Congenital plasma prekallikrein deficiency prolongs the aPTT but is not associated with spontaneous bleeding in humans or animal models, distinguishing prekallikrein from coagulation factors required for hemostasis.

• Emerging flow-based and mechanobiological insights into arterial thrombosis highlight the central role of contact pathway activation under pathological high-shear conditions, positioning prekallikrein as a clinically relevant antithrombotic target.

• Antisense oligonucleotide and small molecule inhibitor studies demonstrate context-dependent roles for prekallikrein in venous and arterial thrombosis models, with therapeutic efficacy achievable without compromising normal hemostasis.

• Clinical validation of prekallikrein-targeting therapies in hereditary angioedema, alongside emerging quantitative biomarker frameworks in vascular disease, supports prekallikrein inhibition as a promising and measurable therapeutic strategy warranting further investigation.