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Semin Thromb Hemost
DOI: 10.1055/a-2681-6784
Letter to the Editor

Resistance or Resilience? Hemostatic Balance in an FV Leiden Elite Athlete

Ciro Miele‡   ‡
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
2   CEINGE Advanced Biotechnologies Franco Salvatore, Naples, Italy
,
Cristina Mennitti‡   ‡
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
,
Alessandro Gentile
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
,
Mariella Calvanese
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
,
Luca Manfredi
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
3   Department of Integrated Activity of Laboratory Medicine and Transfusion, University of Naples “Federico II,” Naples, Italy
,
Andrea Ruggiero
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
,
Immacolata Randa
3   Department of Integrated Activity of Laboratory Medicine and Transfusion, University of Naples “Federico II,” Naples, Italy
,
Ferdinando Cirillo
5   Department of Clinical Medicine and Surgery, University of Naples “Federico II”, Naples, Italy
,
Giovanni D'Alicandro
4   Department of Neuroscience and Rehabilitation, Center of Sports Medicine and Disability, AORN, Santobono-Pausillipon, Naples, Italy
,
Nadia Tinto
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
2   CEINGE Advanced Biotechnologies Franco Salvatore, Naples, Italy
3   Department of Integrated Activity of Laboratory Medicine and Transfusion, University of Naples “Federico II,” Naples, Italy
,
Giulia Frisso
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
2   CEINGE Advanced Biotechnologies Franco Salvatore, Naples, Italy
3   Department of Integrated Activity of Laboratory Medicine and Transfusion, University of Naples “Federico II,” Naples, Italy
,
Cristina Mazzaccara
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
2   CEINGE Advanced Biotechnologies Franco Salvatore, Naples, Italy
3   Department of Integrated Activity of Laboratory Medicine and Transfusion, University of Naples “Federico II,” Naples, Italy
,
Olga Scudiero
1   Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II,” Naples, Italy
2   CEINGE Advanced Biotechnologies Franco Salvatore, Naples, Italy
3   Department of Integrated Activity of Laboratory Medicine and Transfusion, University of Naples “Federico II,” Naples, Italy
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While physical inactivity is an established risk factor for venous thromboembolism (VTE), it might seem logical to assume that an active, athletic lifestyle would offer protection against it.[1] Exercise, indeed, plays a crucial role in preventing numerous chronic conditions, including cardiovascular disease, diabetes, and obesity.[2] [3] However, intense training can temporarily disrupt the hemostatic system, upregulating prothrombotic pathways and platelet aggregation, which may lead to the formation of blood clots.[4]

Through a tightly coordinated series of enzymatic reactions, hemostasis preserves the dynamic balance between procoagulant activity and anticoagulant control. This ensures the fluidity of blood and its containment within the circulatory system.

Both congenital and acquired alterations in the coagulation system can lead to thrombotic events when the procoagulant forces predominate, or hemorrhagic events if the factors promoting thrombus formation are dysfunctional or absent.[5] [6] Despite the significant impact of genetic defects, acquired coagulopathies, due to their numerous clinical consequences, are equally critical, representing some of the most challenging clinical conditions to define. VTE, which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), represents one of the most common vascular conditions worldwide. VTE incidence has shown an upward trend in recent years, affecting 1 to 2 per 1,000 adults annually.[7] VTE can be triggered through a complex interaction of factors, including blood stasis, damage to blood vessel linings, and hypercoagulability, known collectively as Virchow's triad.[8] Intense physical activity, combined with factors such as dehydration, injuries, prolonged travel, and the use of performance-enhancing supplements, such as anabolic steroids and erythropoiesis-stimulating agents (ESAs), can further increase the likelihood of thrombotic events, as these substances affect various aspects of the hemostasis system, creating an environment in otherwise healthy athletes where blood clots are more likely to form.[9]

This risk is further increased in athletes with a genetic predisposition to thrombotic disorders.

Hereditary risk factors for VTE include deficiencies in glycoproteins involved in the natural anticoagulant system—such as antithrombin (AT), protein C (PC), and protein S (PS)—as well as dysfibrinogenemia, factor V Leiden (FV Leiden), and the prothrombin G20210A mutation. In particular, FV Leiden is a genetic prothrombotic condition that induces resistance to activated protein C, thereby impairing the regulation of thrombin generation. PC in its active form, along with its cofactor PS, promotes the inactivation of factors Va (FVa) and VIIIa (FVIIIa) through arginine (R) residues, thus triggering a physiological anticoagulant mechanism.

In FV Leiden, a missense mutation in the factor V (FV) gene at G1691A results in R506 being switched to glutamine (R506Q), which alters the cleavage site on factor Va for activated protein C (APC), creating a pro-thrombotic environment by minimizing the degradation rate of FVa, and prolonging prothrombinase complex activity.[10] [11] Considering the significant prevalence of FV Leiden, estimated at 5 to 10% in the general population in Europe, the accurate identification of this phenotype is clinically essential.[12] Currently, the majority of commercially available assays for detecting APC resistance (APCR) employ a modified partial thromboplastin time–based or prothrombin time–based factor V tests to evaluate the functional capacity of PC in inactivating factor Va.[13] [14] The degree of clotting time prolongation after APC addition, compared with the baseline clotting time without APC, serves as an index for in vitro APC resistance.[15] Besides the classical coagulation assays, a new generation of global coagulation tests, called thrombin generation assays (TGAs), has been developed to simultaneously and continuously measure thrombin generation and its inhibition, thus providing information on the dynamics of clot formation resulting from the action of both procoagulant and anticoagulant drivers. Here, we describe the case of an elite basketball player positive for APCR test, using a functional phenotypic assay, but with a thrombogram profile, obtained with ST Genesia TGA, showing an unexpectedly positive response to thrombomodulin (TM) upon its addition to the system.

A 20-year-old elite athlete, a part of the basketball team supervised by our team for approximately 5 years, was referred to our department at the University Hospital Federico II of Naples (Italy) for a routine laboratory check-up. Given the correlation between thrombotic events and extreme physical activity, blood tests included thrombophilia screening to assess both acquired and inherited risk factors, which revealed a positive result for APCR using a functional clotting-based assay. He denied any personal history of cardiovascular or pulmonary conditions and was unaware of any family history of PE, DVT, or genetic risk factors for thrombophilia. No medications were reported.

First-level coagulation tests, such as activated partial thromboplastin time (aPTT), prothrombin time (PT), and Clauss fibrinogen, and second-level hemostasis assays used for thrombophilia screening, including natural inhibitors of clotting factors (PC, PS, and AT), APCR, lupus anticoagulant, and homocysteine, were measured using the automated ACL TOP 550 coagulometer (Instrumentation Laboratory Company, Bedford, MA, USA), with dedicated HemosIL reagents (Instrumentation Laboratory Company, Bedford, MA, USA). Anti-cardiolipin antibodies (aCL-IgG/IgM), anti-β2-glycoprotein I antibodies (aβ2GPI-IgG/IgM), and von Willebrand factor antigen (VWF:Ag) were tested by chemiluminescence immunoassay using HemosIL AcuStar (Instrumentation Laboratory Company, Bedford, MA, USA). Global assessment of coagulation was investigated in a thawed platelet-poor plasma (PPP) through TGA employing the fully automated ST Genesia (STG, Stago, Asnières-sur-Seine, France). Thrombin generation was assessed using the STG-ThromboScreen (STG-TS) reagent kit, containing a mixture of phospholipids and a medium picomolar concentration of human tissue factor (TF), in the presence and absence of a rabbit-derived TM.

The following TG parameters with and without TM were recorded: lag time, representing the time it takes for thrombin to begin forming after the initiation of coagulation; peak height, which is the maximum amount or concentration of thrombin that is generated during the assay; time to peak, the time needed to reach the peak; velocity index, the speed needed to reach the peak of thrombin generated; endogenous thrombin potential (ETP), describing the area under the thrombogram curve, which corresponds to the net amount of thrombin produced; and TM-induced ETP inhibition, evaluating the inhibition or suppression of ETP mediated by TM.[16] The latter is calculated by the instrument software using the formula (ETP without TM – ETP with TM)/(ETP without TM). Each plasma sample analyzed was run in parallel with a normal human plasma, STG-RefPlasma, and a normalized result for each patient was obtained automatically by the instrument's program. All procedures took place according to the manufacturer's recommendations. Thrombophilia evaluation ([Table 1]) highlighted APCR positivity (0.68 normalized ratio [NTR], reference values >0.75), which is an aPTT-based functional assay that measures the ability of protein C to inactivate factor Va, assessed using HemosIL Factor V Leiden reagent (Instrumentation Laboratory Company, Bedford, MA, USA). APCR was later confirmed by PCR analysis, which detected a heterozygous FV Leiden mutation. The prothrombin 20210 mutation and congenital or acquired deficiencies of natural coagulation inhibitors (encompassing AT, PC, and PS) were ruled out beforehand to ensure no additional interference with the TGA. Noteworthy was an increase in free PS levels which, although not associated with thrombotic risk, is the only parameter that, together with APC, fell outside the reference values.

Table 1

Laboratory hemostatic features of the athlete

Parameters

Results

PT

(r.v. 0.8–1.20 ratio)[a]

1.07

aPTT

(r.v. 0.8–1.20 ratio)[a]

1.12

Fibrinogen

(r.v. 160–350 mg/dL)[a]

257

D-Dimer

(r.v. 0–500 ng/mL)[a]

239

Antithrombin

(r.v. 70–120%)[a]

109

Protein C

(r.v. 70–120%)[a]

98

Free protein S

(r.v. 72–123%)[a]

186

Lupus anticoagulant

(r.v. 0.8–1.20 ratio)[a]

1.12

Anti-cardiolipin IgG

(r.v. <20 U/mL)[a]

2.2

Anti-cardiolipin IgM

(r.v. <20 U/mL)[a]

1.6

Anti-β2 glycoprotein I IgG

(r.v. <20 U/mL)[a]

4.5

Anti-β2 glycoprotein I IgM

(r.v. <20 U/mL)[a]

0.6

APCR

(r.v. >0.75 NTR)[a]

0.68

FVIII

(r.v. 50–130%)[a]

116

VWF:Ag

(r.v. 63–159%)[a]

153

Abbreviations: APCR, activated protein C resistance; aPTT, activated partial thromboplastin time; FVIII, factor VIII; PT, prothrombin time; VWF:Ag, von Willebrand factor antigen.


a Reference values (r.v.) were defined based on healthy population. Bold values represent values outside the r.v.


Thrombin generation was measured in a PPP sample thawed at 37°C for 2 to 3 minutes, using the automated ST Genesia analyzer with the STG-ThromboScreen kit following the manufacturer's instructions.

As described in [Table 2], thrombogram parameters obtained from the ST Genesia software with and without TM ([Fig. 1]) highlighted an increase in the peak height, the ETP, and the velocity index. The addition of TM to the athlete's plasma, which serves as an endogenous anticoagulant protein, underlined an unexpected efficiency of the physiological coagulation inhibition system despite a positive APCR test, as highlighted by the ETP inhibition value obtained in our study (46.8%; reference values 43–66%). These data are of considerable importance and emphasize the effectiveness of the athlete's anticoagulation system, despite the factor V Leiden mutation.

Table 2

Thrombin generation results from the athlete's plasma using the ThromboScreen reagent with and without TM

TGA PARAMETERS

RESULTS

Lag Time

(r.v. 2.2-2.8) [a]

2.52

Peak Height

(r.v. 169-219) [a]

266.7

Time To Peak

(r.v. 5.1-6.4) [a]

5.37

ETP

(r.v. 974-1362)[a]

1693

Velocity Index (nM/min)

(r.v. 59.6-95.2) [a]

122

Start Tail (min)

(r.v. 14.9-19.3) [a]

19.01

ETP Inh. (%)

(r.v. 43-66)[a]

46.82

Abbreviations: ETP, endogenous thrombin potential; ETP Inh., endogenous thrombin potential inhibition.


a Reference values (r.v.) obtained by Ninivaggi et al.26 Bold values represent values above the r.v.


Zoom
Fig. 1 Thrombogram curves obtained from the athlete's plasma using ST-Genesia. (A) Thrombin generation assay (TGA) curve measured in the absence of thrombomodulin (TM). TGA parameters are related to a reference plasma and expressed as ratios (temporal data) or percentages (thrombin concentration–related data). (B) TGA curve measured in the presence of TM.

It has been widely demonstrated how extreme physical activity leads to an increased hypercoagulable state in sportspeople.[9] [17] The thrombotic risk in this group of people is further enhanced when acquired conditions are combined with predisposing genetic factors (e.g., FV Leiden and prothrombin G20210A mutations).

Traditional coagulation assays, such as PT and aPTT, although reflecting a measure of thrombin generation, do not accurately show the true balance of coagulation that occurs in vivo, being prolonged when using plasma from individuals with deficiency in procoagulant factors, but remaining within normal ranges in congenital deficiencies of PC, PS, or AT.[18] This creates a paradox, since patients with deficiencies in natural anticoagulant inhibitors would typically be expected to have shorter PT and aPTT values due to their increased thrombin production compared with healthy individuals.[19] Considering thrombin's unique role in blood clotting, the propensity of a plasma sample to produce thrombin, utilizing the TGA, can offer valuable insights into thrombotic or bleeding risk.[19] Thrombin's pivotal role within the sophisticated pathway of coagulation is demonstrated by its dual function as both a procoagulant and an anticoagulant molecule.[20] Its procoagulant effect involves the conversion of fibrinogen into fibrin, platelet activation, and the initiation of a positive feedback loop that promotes further thrombin production upstream.[20] To prevent continuous activation of the coagulation system, thrombin binds to TM on the surface of endothelial cells, triggering the anticoagulant pathway through activation of PC.[20] PC, together with its cofactor, PS, inhibits further thrombin formation via a negative feedback mechanism, achieved through proteolytic degradation of FVa and FVIIIa.[20] Additional stimuli contributing to the downregulation of thrombin generation include tissue factor pathway inhibitor (TFPI) and by AT. For this reason, upon the identification of a positive APCR result in an elite basketball player, to assess its overall tendency to form thrombin, we conducted a comprehensive evaluation of the entire coagulation process through TGA. Thrombograms obtained using a reagent containing phospholipids and an intermediate concentration of TF, with and without the presence of TM, displayed an increase in peak height, ETP, and velocity index, all parameters related to hypercoagulability, but an unexpected inhibition of ETP of almost 50% after the addition of TM, despite the resistance of FV to PC.

Our results confirmed the strong correlation between hemostasis and physical exercise, suggesting that although this relationship can be linked to a transient state of hypercoagulability, it can also promote compensatory mechanisms that enhance the activation of secondary physiological anticoagulation pathways. To date, no studies have directly assessed APCR in response to exercise. Some studies have examined indirect markers linked to the protein C pathway in the context of physical activity.[21] [22] Cerneca et al compared specific hemostatic parameters among elite athletes from endurance sports, such as rowing and running, and strength sport, such as weightlifting. They observed higher basal activities of antithrombin and protein C in marathon runners compared with rowers. A significant decrease in protein C levels has been reported after near-maximal exercise in rowers, whereas no such change was observed in marathon runners or weightlifters. Since weightlifters did not exhibit any immediate alterations in secondary hemostasis parameters following exercise, it is plausible that the type of physical activity performed, along with the associated recovery periods, plays a key role in determining the changes observed in the coagulation cascade.[21]

Recent studies have highlighted that PS, beyond its role as a cofactor for activated protein C, can also exhibit its inhibitory effect on coagulation independently of PC, by acting as a cofactor for TFPI to enhance FXa inhibition and by directly inhibiting FIXa, thereby limiting FXa generation.[23] [24] Specifically, the anticoagulant mechanism mediated by PS and involving TFPI is exerted through the TFPIα isoform, particularly via the binding of its Kunitz domain to PS. This association enhances the rate of FXa inhibition by approximately 4- to 10-fold, thereby increasing TFPI's anticoagulant function.[25]

In this context, given the striking effectiveness of the physiological anticoagulation system observed, as reflected in the increased ETP inhibition value, and the athlete's asymptomatic status, it is plausible to hypothesize that his hemostatic system—likely influenced by the positive effects of physical activity—may have fostered collateral anticoagulation mechanisms, despite the presence of APCR. As a result, anticoagulant therapy was deemed unnecessary, and no further monitoring is planned, as the athlete is no longer under our care.

Another important piece of evidence supporting this hypothesis is the increase in PS levels (186%) observed in this athlete. This elevation, which markedly exceeds the upper reference limit of 123%, could be linked to the previously mentioned mechanism of PC-independent PS inhibition, potentially contributing to the efficiency of the anticoagulation system and, to some extent, compensating for the primary defect associated with the FV Leiden mutation.

These hypotheses should be confirmed in athletes with similar conditions, but they may provide a basis for further exploration of the mechanisms underlying the APC-independent anticoagulant activity of protein S.

‡ These authors contributed equally to this work.




Publication History

Received: 06 April 2025

Accepted: 12 August 2025

Article published online:
25 August 2025

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