Investigations on the Hemostatic Potential of Physiological Body Fluids

Abstract

Current blood coagulation models consider the interactions between blood, the vessel wall, and other tissues that expose tissue factor (TF), the main initiator of coagulation. A potential role of body fluids other than blood is generally not considered. In this review, we summarize the evidence that body fluids such as mother's milk saliva, urine, semen, and amniotic fluid trigger coagulation. The ability of these body fluids to trigger coagulation is explained by the presence of extracellular vesicles (EVs). These EVs expose extrinsic tenase complexes (i.e., complexes of TF and activated factor VII) that can trigger coagulation. Why these body fluids share this activity, however, is unknown. Possible explanations are that these body fluids contribute to hemostatic protection and/or to the regulation of the epithelial barrier function. Further investigations may help understand the underlying cellular and biochemical pathways regulating or contributing to coagulation and innate immunity, which may be directly relevant to medical conditions such as gastrointestinal bleeding and chronic inflammatory bowel disease.

Keywords

tissue factor - factor VIIa - extracellular vesicles - physiological body fluids

Introduction

Hemostasis is the process that stops the bleeding of damaged vessels and encompasses the tightly regulated processes of blood clotting, platelet activation and aggregation, and vascular repair.[1] Upon tissue damage, extravascular tissue factor (TF, a transmembrane glycoprotein) acts as a receptor for blood-borne coagulation factor (F) VII (FVII), which leads to activation of FVII to FVIIa. The complex of TF and FVIIa is known as the extrinsic tenase complex. This complex activates FX and FIX. In a waterfall cascade, additional macromolecular complexes (such as the prothrombinase complexes of FXa and FVa and the intrinsic tenase complexes of FVIIIa and FIXa) are formed, which leads to the formation of a fibrin clot and platelet activation via thrombin. Activated platelets not only stabilize the fibrin clot but also accelerate coagulation.

Current models of blood coagulation consider the interaction between blood, the vessel wall, and other extravascular TF-exposing tissues.[1] [2] [3] A potential role of body fluids other than blood is not regarded in these models. In this review, we summarize the evidence that physiological body fluids can contribute to hemostasis and may be involved in the regulation of the epithelial barrier function. Body fluids are discussed in this review according to their main physiological functions in five sections: (1) alimentation (mother's milk), (2) digestion (saliva, pancreatic juice, and bile), (3) homeostasis (lymph, urine, bile, tears, and cerebrospinal fluid), (4) reproduction (semen, follicular fluid), and (5) development (amniotic fluid).

Alimentation

Mother's Milk

In the 1930s, hemophilia was a devastating hereditary condition with a mean life expectancy of ∼8 years,[4] but an effective factor substitution therapy for patients with hemophilia was not discovered until the 1960s.[5] What could a doctor do in the 1930s when facing unstoppable bleeding in boys with hemophilia? Alphons Solé, an Austrian pediatrician, came up with the idea (translated from German) “to mix the two human body fluids that have the ability to clot,” namely, mother's milk and blood, based on his heuristic thought that “natural remedies may be present in organs and fluids of the human organism.”[6] Strikingly, Solé observed that even small amounts (“droplets”) of mother's milk induce immediate clotting of blood. Next, Solé added mother's milk to “hemophilia blood” and observed “a coagulant effect that surpasses by far all other known hemostatic agents.” Inferring that his observations may be useful in treating external bleeding in hemophiliacs, Solé did not hesitate to translate his experimental findings into clinical practice, and therefore he meticulously planned what is called today an interventional clinical trial.

The main treatment procedures of his trial were laid out in his publication: “(1) Use mother's milk from healthy woman. (2) Wash the breast with soap and water and then disinfect the breast with alcohol. Express the mother's milk immediately before its use and discharge the first milliliters. (3) Soak iodoform tamponades in the freshly collected mother's milk and apply it to the bleeding spot. Take care that the tamponade does not slip out of position with auxiliary devices. (4) Keep the mother's milk–soaked tamponades as long as possible and remove them carefully, because removal may lead to anew bleeding.” Solé reported the observations from his trial by carefully describing the effects of therapeutic administration of mother's milk–soaked tamponades on severe external bleeding from five hemophiliacs. These cases, which all had a favorable outcome, are summarized in [Table 1].

Table 1 Management of external bleeding with mother's milk–soaked tamponades in male persons with hemophilia as reported by Alphons Solé in 1934

	Clinical history	Previous treatments	Reason for consultation of Alphons Solé	Laboratory results	Current treatment	Outcome
Case 1: 14-year-old adolescent	Life-threatening bleeding after tooth extraction at the age of 12	None	Planned extraction of 2 molar teeth	Whole blood clotting time of 26 minutes, normal platelet count	After extraction of the two molar teeth, the wounds were tamponaded with mother's milk–soaked pads, which were kept in position with a rubber prosthesis	Cessation of postsurgical bleeding, 2 days later minimal bleeding during the change of the tamponade, then no further bleeding occurred and the wound healed under the tamponade
Case 2: 4-year-old boy	Unusual hematomas and bleeding from an early age after minor traumas	Cauterization of bleeding wounds, IM injection of horse serum, Nateina,[a] calcium, gelatin	Ongoing bleeding after laceration on the forehead	Whole blood clotting time of 12 hours, normal platelet count	A mother's milk–soaked tamponade was applied at the wound site	Cessation of bleeding, minimal bleeding during the change of the tamponade, then no further bleeding
Case 3: 9-year-old boy	Regular hospitalizations due to bleeding of the joints and internal bleeding	Information not provided	Ongoing bleeding after broken glass injury of the Sole	Whole blood clotting time of 1 hour and 15 minutes, normal platelet count	The wound was incised 4 cm wide and 2 cm deep and plugged with a mother's milk–soaked tamponade	Cessation of bleeding, after 2 days, minimal bleeding during the change of the tamponade, the tamponade was kept for an additional 9 days, and only parts were removed until complete healing of the wound
Case 4: 24-year-old man	No apparent bleeding tendency before current bleeding, uncle was diagnosed with hemophilia	None	Ongoing bleeding after submucosal resection of septum nasi and turbinates	Information not provided	Mother's milk–soaked tamponades were applied to the nostrils after 6 days of recurrent severe bleeding with usual tamponades	Cessation of bleeding, no bleeding after removal of tamponade 2 days later, 6 days later single minimal nosebleed, then no further bleeding
Case 5: 11-year-old boy	Severe hemophilia with recurrent joint bleeding, hematomas, contractions	Information not provided	Ongoing bleeding due to loosened primary tooth	Information not provided	Mother's milk–soaked tamponade applied to the bleeding spot	Cessation of bleeding, minimal bleeding the next day during the change of the tamponade, then no further bleeding

^a Nateina is the term for a mixture of vitamins A, B, C, and D, calcium phosphate, and milk sugar, which was believed to have hemostatic properties.

Kraszewski and Lindenfeld confirmed Solé's observations on the coagulant potential of mother's milk in 1936.[7] In cow's, goat's, and mare's milk, they found (translated from German) “very limited coagulant potential” and they excluded thrombin being the active coagulant component in mother's milk “because addition of mother's milk to fibrinogen concentrates did not generate fibrin.” They observed that mother's milk converts prothrombin to thrombin, and resembles what they called cytozyme. The term cytozyme was used in the 1930s, which is even before the terms thromboplastin and thrombokinase were introduced, to describe a highly coagulant crude homogenate of human or animal brain or placenta, which was used as a laboratory reagent to induce clotting.[8] To date, we know that the active component of cytozyme is TF, a receptor for FVII, which promotes (auto) activation of FVII to FVIIa. This process requires the presence of negatively charged phospholipids, which are also present in these homogenates.[9] Like Alphons Solé, Kraszewski used tamponades soaked in mother's milk to stop external bleeding. As a surgeon, Kraszewski applied mother's milk–soaked tamponades on wound sites after prostatectomies and rhinopharyngeal surgeries. He reported, again not unlike Solé, that the hemostatic effect of these tamponades “by far exceeds all other hemostats that are currently used in the clinics.”[8]

Based on the observations of Solé, the Swiss pediatrician Eduard Glanzmann recommended in one of his lectures (translated from German): “Put a tamponade soaked with mother's milk and press it on the bleeding spot!” in 1934.[10]

In 1961, M. W. Hess concluded that (translated from German) “no coagulation factors could be found, apart from a tissue-kinase-type thromboplastic activity, which interfered in all the determinations.”[11] After 1961, no new articles were published on the coagulant properties of mother's milk for the next 60 years.

In 2021, we showed that the coagulant potential of mother's milk is explained by the presence of extracellular vesicles (EVs) exposing TF.[12] In the same publication, cow milk completely lacked coagulant potential. In 2022, we demonstrated that EVs in mother's milk expose not only TF but also the extrinsic tenase complex of TF and FVIIa.[13] In vitro coincubation of mother's milk and simulated gastric and pancreatic fluid confirmed that the coagulant potential of mother's milk can survive digestive conditions as encountered in infants' gastrointestinal systems. Therefore, we concluded that the coagulant potential of mother's milk likely is present also within the gastrointestinal system of breastfed infants. One explanation for the coagulant potential of mother's milk could be the occurrence of nipple skin damage in most breastfeeding women. Milk-derived EVs may seal these wounds and thereby prevent pathogen invasion. Another explanation for the coagulant potential of mother's milk may be vitamin K deficiency at birth, which predisposes newborns to vitamin K deficiency bleeding.[14]

Digestion

Saliva

Saliva is produced by salivary glands and contributes to the digestion of food and the maintenance of oral hygiene.[15] In 1928, the English surgeon John B. Hunter reported that human saliva triggers blood clotting.[16] In 1932, his observation was confirmed by Bellis and Scott, who also investigated the effect of saliva on blood clotting in persons with hemophilia.[17] [18] They reported that the blood clotting time of hemophilia patients shortened from 2 hours to a few minutes with the addition of autologous saliva. In 1938, Glazko and Greenberg reported that the effect of saliva on blood coagulation resembles that of a thromboplastin,[19] which is consistent with the findings reported by Solé.[6] In 1957, Nour-Eldin and Wilkinson investigated the effect of normal saliva in plasma from persons with hemophilia A (i.e., FVIII deficiency), hemophilia B (FIX deficiency), and FVII deficiency.[20] Saliva shortened the clotting time of FVIII- and FIX-deficient blood, showing that saliva compensates for the lack of coagulation amplification via intrinsic tenase complexes of FVIIIa and FIXa, when contacting blood. Interestingly, normal human saliva also shortened the clotting time of FVII-deficient plasma.[20] This was surprising because this indicates that not only “a thromboplastin” is present in saliva, but also FVII(a) (i.e., the ligand of TF). Indeed, in 2022, we confirmed that complexes of TF and FVIIa are present in saliva.[13] These complexes are present on salivary EVs, which may be involved in efficiently separating the “milieu exterieur” and “milieu interieur” by promoting hemostasis, thereby not only reducing blood loss but also the risk of infection by preventing pathogen invasion.

Pancreatic Juice

Pancreatic juice is a product of the exocrine pancreatic glands, containing digestive enzymes.[21] In the 1930s, it was reported that trypsin, a pancreatic protease, shortens the clotting of blood.[22] [23] In 1973, it was shown that trypsin converts prothrombin to thrombin.[24] There is a structural and functional homology between proteases of the digestive system (i.e., trypsin and chymotrypsin) and proteases of the coagulation system (FXII, FXI, FIX, FX, FVII). The latter are therefore also known as trypsin-like serine proteases.[3] Whether this is of clinical relevance, for example, in the prothrombotic state that is associated with acute pancreatitis, remains to be investigated.

Bile

Bile is a yellowish-green fluid produced by the liver that promotes digestion of lipids in the small intestine.[25] Bile completely inhibits the clotting of blood because bile salts are natural detergents that dissolve phospholipids, which are essential in the blood coagulation system and provide a surface for the aggregation of coagulation factors.[26] [27]

Homeostasis

Lymph

Lymph is a clear to milky fluid that passes from intercellular spaces of body tissues to lymphatic vessels and enters the blood via the thoracic duct.[28] To the best of our knowledge, the hemostatic effects of lymph have not been investigated so far experimentally. However, lymph physiologically drains into the blood circulatory system, and this does not lead to coagulation activation. Thus, based on this observation and the fact that blood from healthy humans does not contain detectable levels of TF indicate that the presence of strong procoagulant components in lymph is unlikely. Still, it has been shown that most components of the coagulation system are present in lymph albeit at significantly lower concentrations than in plasma.[29] [30]

Urine

Urine is a liquid secreted by the kidneys, which is rich in end-products of protein metabolism and salts.[31] In 1935, Wilhelm Grunke reported that a few droplets of urine induce the clotting of hemophilic blood (type of hemophilia was not specified).[32] The urine from persons with hemophilia had the same effect, a finding we reproduced recently.[33] Since the coagulant properties of urine were heat sensitive, Grunke concluded that urine must contain a thrombokinase (i.e., an enzyme that triggers blood clotting).[32] In 1947, Tocantins and Linsquist observed that hemophiliacs (again hemophilia type was not further specified) not only frequently have hematuria but also experience severe attacks of renal colics and passing of thin clots via their urine, which seems in marked contrast with the prolonged blood clotting times of hemophiliacs.[34] As Tocantins and Linsquist knew about the presence of “a thromboplastic substance” in tissues,[35] saliva,[16] and mother's milk[6] (but, as it seems, were unaware of Grunke's pioneering investigations), they concluded that a thromboplastin may be present in the urine of hemophiliacs. After showing that dialyzed and lyophilized urine can be used as a coagulant in vitro, and against all ethical reasoning and against the Hippocratic Oath (which they must have taken as medical doctors), in vivo experiments were performed in rabbits, dogs, and humans.[34] First, Tocantins and Linsquist injected reconstituted lyophilized urine at 60 mg/kg body weight intravenously into rabbits, which developed temporarily a cardiorespiratory shock, from which the rabbits recovered. In dogs, intravenous injection of a lower concentration had a moderate transitory effect on blood pressure. Next, they injected reconstituted urine into persons with hemophilia A. Administration of reconstituted lyophilized urine at a rate of 5 to 10 mg/minute was “well tolerated in most patients” (more detailed information was not provided), but when this rate was increased, symptoms including “flushing, pounding behind the eyes and in the abdomen, and headage” occurred frequently. Intravenous injection of reconstituted lyophilized urine shortened the clotting time of hemophilic blood and this effect was dose-dependent.

An ethically more sound approach was chosen by Vonkaulla and Vonkaulla in 1963 to treat bleeding in persons with acquired hemophilia.[36] Acquired hemophilia is an autoimmune disorder, in which autoantibodies neutralize coagulation factors, often FVIII.[37] First, they isolated and lyophilized the coagulant fraction of pooled normal urine. Subsequently, they added the coagulant urine lyophilizate to sterile resorbable gelatin sponges and topically applied these sponges in 25 patients with circulating inhibitors and continuous bleeding from external wounds. The hemostatic effect of these topically applied sponges was described as excellent, very similar to the previously mentioned hemostatic effect of mother's milk–soaked tamponades as applied by Alphons Solé.[6]

Wiggins et al showed that the coagulant potential of urine is associated with 0.1 to 1 µm lipid vesicles,[38] which according to recent nomenclature are now called EVs.[39] The EV-associated coagulant potential of urine was also present in FVII-deficient plasma, and therefore Wiggins et al concluded that “this microvesicular procoagulant activity was mostly factor-VII like as judged by clotting assay using human factor-deficient plasmas.”[38] Unaware of this article, we confirmed this finding recently.[13] Wiggins et al also performed ultrastructural studies in rabbit kidneys and demonstrated the presence of EVs in the proximal tubular lumen, EV budding from glomerular epithelial cells, and adjacent fibrin deposition.[38] These findings all point to the kidney as the source of coagulant urinary EVs rather than the urothelium. The suspected presence of TF in urine was confirmed in the following years, because the coagulant potential of urine was completely inhibited by a monoclonal antibody against human TF.[40] [41] Labeling of urinary EVs with annexin V confirmed the exposure of negatively charged phospholipids that are required for binding of coagulation factors essential for coagulation.[42] Urinary EV-TF activity was also investigated in different patient groups and elevated levels were reported in patients with colon, breast, prostate, and bladder cancer compared with healthy controls.[41] [43] Carty et al discussed the potential source of elevated urinary TF activity in cancer patients and concluded that a “spillover” from peripheral blood is unlikely because the molecular weight of TF is too large to allow filtration by the undamaged glomerulus.[41] The precise source of elevated urinary EV-TF activity in cancer patients, therefore, remains unclear.

Tears

Tear fluid or lacrima is a clear liquid that is secreted by the lacrimal glands of all land mammals.[44] The coagulant potential of tear fluid has been investigated by Liu et al in 2022.[45] They isolated and characterized EVs from tear fluid and demonstrated the presence of TF-exposing EVs, which shorten the clotting time of EV-depleted plasma in a TF-dependent manner. The presence of TF/FVII(a)-complex–exposing EVs in tears seems likely, considering that these EVs have been detected in most other coagulant physiological body fluids such as milk, saliva, urine, and amniotic fluid.

Cerebrospinal Fluid

Evidence indicates that normal cerebrospinal fluid has no or at best very limited hemostatic potential.[46] [47] [48]

Reproduction

Semen

Semen is a male bodily fluid that contains spermatozoa that fertilize the female ovum in the reproduction process.[49] In 1942, Huggins and Neal demonstrated that human semen triggers coagulation, as it reduced the clotting time of recalcified plasma at dilutions up to 10,000-fold.[50] More than five decades later, in 1997, Fernández et al reported that the potent coagulant potential of human semen is due to the presence of TF that is bound to prostasomes.[51] The term “prostasomes” has been coined to describe the presence of small vesicles in prostate secretions, which originate from acinar epithelial cells of the prostate.[52] In this review, we will stick with the umbrella term EVs instead of prostasomes, because the term EVs is more comprising, as it includes vesicles of diverse cellular origin as well as vesicles from different routes of biogenesis. To prove the presence of coagulant TF on seminal EVs (i.e., prostasomes), Fernández et al used both functional coagulation assays and confirmed the presence of TF on EVs using electron microscopy.[51] Fernández et al showed that seminal fluid induces the clotting of FVII-deficient plasma, and this coagulant potential was completely blocked with an antibody against FVII, which points again to the presence of the TF/FVIIa complex on EVs in semen.[51] Carson and De Jonge,[53] and a few years later Lwaleed et al,[54] confirmed the presence of FVII(a) in semen and showed that in semen FVII(a) forms extrinsic tenase complexes together with TF. Lwaleed et al found low overall levels of FVII in semen (∼4% of the plasma concentration), whereas levels of FVIIa (i.e., the functional fraction of FVII) were comparably high in semen as in plasma (∼0.2 nM).[54]

Follicular Fluid

Follicular fluid is a liquid surrounding the ovum. This fluid originates mainly from granulosa cells of the ovarian follicle.[55] Gentry et al investigated the presence of coagulation factors in follicular fluid.[56] Applying functional assays and Western blots, they found that prothrombin, FVII, and FX occur in follicular fluid at concentrations similar to plasma levels of healthy adults. Low levels of FV were detected and other coagulation factors including FVIII and FIX were not detectable. TF was not detected in follicular fluid, and follicular fluid does not trigger the clotting of blood or plasma.[57] Interestingly, FVII is present in follicular fluid at high levels[56] and it remains to be investigated whether this plays a role in ontogenesis.

Development

Amniotic Fluid

Amniotic fluid is a clear liquid that surrounds and protects the fetus during pregnancy.[58] In 1926, Ricardo Meyer first described amniotic fluid embolism as a major cause of obstetric shock and maternal death.[59] Following amniotic fluid embolism, activation of the coagulation system is observed, which frequently leads to overt disseminated intravascular coagulation.[60] The presence of a thromboplastin-like coagulant in amniotic fluid was first described by Weiner et al in 1949,[61] who reported that amniotic fluid shortens the clotting time of recalcified plasma, as well as blood from healthy individuals and from persons with hemophilia, which confirms aforementioned investigations on the hemostatic potential of other physiological body fluids.

In 1972, Phillips and Davidson performed a more detailed analysis of the coagulant potential of amniotic fluid, adding amniotic fluid to coagulation factor-deficient plasma.[62] Amniotic fluid induced clotting of plasma deficient in coagulation factors of the intrinsic system (i.e., FVIII-, FIX-, and FXI-deficient plasma). Amniotic fluid was also coagulant in FVII-deficient plasma (similar to milk, saliva, urine, and semen). Phillips and Davidson concluded that amniotic fluid “is an activator of factor X and may function in a manner that is similar to Russell's viper venom.”[62] Lockwood et al investigated the coagulant potential of amniotic fluid in 1991 and they were the first to report that TF is present in amniotic fluid.[63] [64] While they did not use the term ”EVs,” they stated that “all amniotic fluid tissue factor appears to be membrane bound, because the ultracentrifuged supernatants contained negligible quantities of tissue factor antigen and activity.” Only very recently we discovered that EVs in amniotic fluid expose extrinsic tenase complexes.[13] In addition, amniotic fluid also contains FVII(a) that is not associated with EVs.[65] This soluble FVII(a) may bind TF that is exposed to the fetal epidermis to regulate its epithelial barrier function.

Looking Back to Look Forward

The hemostatic potential of physiological body fluids other than blood has been intensively studied over the last century. Most investigations were performed in the 1930s and 1940s, then to a lesser extent studies were performed until the 1980s, and, finally, the knowledge about the hemostatic potential of body fluids seems to have been almost completely forgotten. For example, Solé's finding that application of mother's milk–soaked tamponades to wounds stops external bleeding in persons with hemophilia was heralded as “the cure of hemophilia” in print media in 1934 ([Fig. 3]), but an English language internet search in 2019 yielded no result (J.T., personal investigation). This only changed when we published on this topic in 2020.[12] Moreover, results from studies investigating the hemostatic potential of body fluids were never summarized. To the best of our knowledge, this is the first review on the topic. Summing up the large body of available data in the current review, we feel that particularly three points should be emphasized.

First, most results from studies that are included in this review are well comparable, because comparable mixing studies of plasma and body fluids were performed. For example, the ability of milk, saliva, urine, semen, and amniotic fluid to induce the clotting of FVII-deficient plasma was identified by independent research groups mixing these body fluids with recalcified FVII-deficient plasma in studies performed between 1957 and 2022[13] [20] [53] [62] [66] [67] ([Table 2], [Figs. 1] and [2]). We only recently identified the presence of TF/FVII(a) complex present on EVs as the underlying cause explaining this coagulant potential.[13]

Table 2 The reported hemostatic potential of physiological body fluids

		Specific hemostatic trigger		Ref.
Body fluid	Reported hemostatic potential	TF/FVIIa-complex–exposing EVs	Other
Amniotic fluid	++	⊕	⊖	13, 59–64
Bile	−	⊖	⊖	26, 27
Cerebrospinal fluid	−	⊖	⊖	46–48
Follicular fluid	−	⊖	⊖	55–57
Gastric juice	−	⊖	⊖	79
Lymph	−	⊖	⊖	29,30
Milk	+++	⊕	⊖	6, 7, 10–13
Nasal secretions	ND
Pancreatic juice	+/?	?	trypsin	22, 23
Saliva	++	⊕	⊖	13, 16–20
Semen	+++	⊕	⊖	49–54
Sweat	ND
Tears	+++	⊕/?	⊖	45
Urine	+/+ +	⊕	⊖	32–34, 36, 38–43

Abbreviations: ++ + , strong; ++, moderate; +, weak; −, negative; ?, unknown; ND, not determined; EVs, extracellular vesicles; FVIIa, activated factor VII; TF, tissue factor; ⊕, present; ⊖, absent.

Fig. 1 Body fluids initiate coagulation of normal (A) and factor VII–deficient (B) plasma. Mother's milk, amniotic fluid, saliva, and urine (n = 5) induce clotting of normal plasma (A) and factor VII–deficient plasma (B) at a final concentration of 5% (vol/vol) in a clotting assay performed as described previously.[13]

Fig. 2 Tissue factor (TF)/activated factor VII (FVIIa)-complex–exposing extracellular vesicles (EVs) in physiological body fluids. Hemostatic TF/FVII(a)-complex–exposing EVs have been detected in mother's milk, saliva, urine, semen, and amniotic fluid. In tears, only the presence of TF-exposing EVs was investigated and confirmed, but the presence of TF/FVII(a)-complex–exposing EVs seems likely. TF/FVII(a)-complex–exposing EVs may also play a role in the regulation of the epithelial barrier function. This hypothesis, however, is still highly speculative.

Second, the question is why completely different body fluids (such as saliva, breast milk, and semen) share this strong hemostatic potential that can be attributed to TF/FVIIa-complex–exposing EVs. To date, we can only speculate about the answer. One explanation may be hemostatic protection following injury. Body fluids may simply act as endogenous hemostats that can boost blood clotting in case of tissue damage, which, for example, could explain the reflex of wound-licking that is observed in many mammals including humans.[68] This explanation (i.e., the function of body fluids as endogenous hemostats) may apply to body fluids such as amniotic fluid, milk, saliva, and urine. However, we believe that this cannot explain, for example, the very strong hemostatic potential of semen,[50] because the male genitourinary tract and particularly the glans penis, which has a robust epithelium, are not prone to bleed.[69] Alternatively, Lwaleed et al suggested an important role of TF and FVIIa in the spontaneous coagulation of seminal plasma, which is observed after ejaculation.[70] [71] This, however, was never further investigated. Another explanation could be a role for TF/FVIIa-complex–exposing EVs in signaling. TF/FVIIa complexes not only cleave FX and FIX, but they can also indirectly induce the cleavage of protease-activated receptor-2 (PAR2) via transactivation of the membrane-anchored serine protease matriptase. PAR2 is a seven-transmembrane-spanning receptor, and cleavage of PAR2 by the TF/FVIIa complex may increase the barrier function of epithelia.[72] Consistently, we recently reported that free soluble FVII(a), which is also present in amniotic fluid besides EVs exposing extrinsic tenase complexes, can bind to TF on keratinocytes. This binding increased the epithelial barrier function of keratinocytes in vitro.[65] We also found that TF is highly expressed in the outermost layer of the fetal epidermis, which contacts amniotic fluid in utero. Therefore, the binding of amniotic FVIIa to TF exposed in the fetal skin may foster skin epithelial barrier function. Another possible cytoprotective mechanism may be the binding of free soluble FVIIa from body fluids to endothelial protein C receptor, which induces signaling via PAR1, which protects against inflammation.[73] [74]

Third, although the research interest in EVs has been growing exponentially, their contribution to physiology is hitherto obscure.[75] [76] Evidence summarized in this comprehensive overview from independent studies points to the involvement of EVs in hemostasis by their exposure of coagulant extrinsic tenase complexes and phospholipids. If so, this might be the first physiological function that can be attributed to EVs.

Finally, the self-reflective question needs to be asked, how much sense it makes trying to spark new interest in the old topic of hemostatic body fluids. Not surprisingly, we believe the answer is “this makes hell of a lot of sense” for several reasons. Most importantly, the unexpectedly high enzymatic activity of TF/FVII(a) complexes in fundamentally different body fluids points to a protective mechanism that is largely unknown so far, and further investigations may improve our understanding of the interplay between coagulation and innate immunity, hence paving the way to new treatment strategies. For example, TF/FVIIa-complex–exposing EVs may increase the gastrointestinal barrier function, which may improve the course of chronic inflammatory bowel disease.[77] Topical application of recombinant FVIIa may bind to TF on the skin of preterm newborns and improve the immature skin barrier function, thereby reducing complications such as skin infections, hypothermia, and dehydration.[78]

In conclusion, limited research on the hemostatic properties of body fluids other than blood has been performed over the last decades. Considering that interactions between TF/VII(a)-complex–exposing EVs from body fluids and epithelia may represent an important protective mechanism that potentially can be reproduced for the treatment of severe medical conditions, this research topic should be revived.[46] [47] [48] [79] [80]

Fig. 3 On November 10, 1934, the Spanish newspaper Diario de Madrid reported: “The cure of hemophilia. The Austrian physician who claims to have cured hemophilia using breast milk. According to the testimony of other doctors, experiments on several patients have given results of undoubted effectiveness.” (With the kind permission of Thomas Solé). Alphons Solé (1901–1983) was a Viennese pediatrician and researcher. Solé fled the Nazi regime in 1937 and set up a children's hospital in Kabul, which he chaired for 10 years. In 1947, he returned to Vienna and chaired the Karolinen Kinderspital.

No conflict of interest has been declared by the author(s).

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Publication History

Received: 11 July 2024

Accepted: 05 August 2024

Article published online:
23 October 2024