What is the clinical spectrum of Thrombocytopenia?
Thrombocytopenia is a hematologic pathology in which counts of platelets (thrombocytes), that intervene in blood clotting and wound healing, are below the lower limit of normal, 150x109/L for adults Santoshi et al., Cureus, Aug 2022, Jinna & Khandar, StatPearls, Jul 2023, Pietras et al., StatPearls, May 2024, Stanley et al., StatPearls, Apr 2023.
Thrombocytopenic patients are at an increased risk of bleeding, thrombosis, and mortality.
Thrombocytopenia prevalence, degree of morbidity, and fatality rate varies according to the disease subtype. The most common cases of thrombocytopenia found in intensive care units are related to drug-induced thrombocytopenia (DIT), sepsis, major bleeding, thrombocytopenic thrombotic purpura (TTP), hemolytic uremic syndrome (HUS), and disseminated intravascular coagulation (DIC).
Decreased platelet production and/or an increased platelet destruction are common features of thrombocytopenia pathophysiology.
Decreased platelet production was reported in conditions such as aplastic anemia (AA) bone marrow failure, drug-related and cancer-related bone marrow suppression, viral infections, systemic conditions such as nutrient deficiency and sepsis, and inherited thrombocytopenia (IT).
Increased platelet destruction is present in primary immune thrombocytopenia (primary ITP), drug-induced ITP, lymphoproliferative disorders, autoimmune conditions like systemic lupus erythematosus, in chronic infections, as well as in several non-immune related conditions.
Thrombocytopenia is directly associated with damage to several organs, including kidneys, brains, heart, liver, and lungs,
What do we know about the etiology of Thrombocytopenia?
Platelets are produced by bone marrow-residing megakaryocytes, and thrombocytopenia is typically linked to dysfunction in bone marrow production.
The causes of thrombocytopenia are very heterogenous and can be internal, external, or a combination of both.
Aplastic anemia (AA) bone marrow failure is an autoimmune disorder that leads to hematopoietic stem and progenitor cell growth inhibition and apoptosis, resulting in pancytopenia - deficiency of all blood cells Giudice & Selleri, Sem. Hematol., Jan 2022. The etiology of AA can be hereditary (Fanconi anemia, Dyskeratosis congenita), toxic exposure (chemotherapy, drugs, radiation) or viral infections (Epstein-Barr virus, cytomegalovirus, hepatitis viruses) Jinna & Khandar, StatPearls, Jul 2023.
Chemotherapy-induced thrombocytopenia (CIT) is a frequent complication of cancer therapies, resulting in increased bleeding risk, dose reduction, and treatment discontinuation that can negatively affect oncologic outcomes Gao et al., Disc. Oncol., Jan 2023. CIT in cancer patients is commonly caused by cytotoxic effects of chemotherapy, damaging hematopoietic stem cells and megakaryocyte progenitors. Beyond bone marrow failure, other mechanisms that underly CIT include immune-mediated platelet destruction, splenic sequestration, and myelophthisis.
Radiation-induced thrombocytopenia (RIT) is also considered as non-immune drug-induced thrombocytopenia, acting through endothelial injury, megakaryocyte apoptosis, and bone marrow suppression Singh & Seed, Int. J. Radiat. Bio., Jun 2017.
The widely used anticoagulant heparin can trigger an abnormal immune response in which the body produces antibodies against platelet factor 4, resulting in heparin-induced thrombocytopenia (HIT) Santoshi et al., Cureus, Aug 2022. HIT can cause life-threating arterial and venal thrombosis, leading to stroke or pulmonary embolism.
Apart from heparin and chemotherapy, other medications associated with drug-induced thrombocytopenia (DIT) act through variable mechanisms, including immune-mediated mechanisms (antibiotics - quinine, vancomycin, sulfonamides, anticonvulsants – carbamazepine, phenytoin), and hematopoietic progenitors DNA synthesis-inhibition mechanisms (antivirals - ganciclovir) Jinna & Khandar, StatPearls, Jul 2023.
Primary immune thrombocytopenia (ITP) is an autoimmune disorder with no identifiable external trigger, characterized by a low platelet count, purpura, and hemorrhagic episodes Pietras et al., StatPearls, May 2024, Jinna & Khandar, StatPearls, Jul 2023, Santoshi et al., Cureus, Aug 2022.
In ITP, platelet destruction involves both humoral (B cell-producing antibody-based) and cellular (CD8 cytotoxic T cell–based) mechanisms.
Platelets are opsonized by B cells that produce endogenous antibodies against platelet glycoproteins, and cleared by macrophages via their antibody-binding Fcy surface receptors. In ITP, an imbalance between Fcy receptors that activate and receptors that inhibit antibody-dependent platelet destruction, plays a role in excessive platelet clearance.
Measurement of Th9 and Th17 cells in active ITP patients and ITP patients in remission, indicated that T helper cells were elevated in disease state, promoting B cell activation, and antibody production Qiao et al., Platelets, Sep 2016.
Secondary ITP typically has an external cause, including drugs, infections, malignancies like chronic lymphocytic leukemia, and autoimmune conditions like systemic lupus erythematosus.
Viral infections, such as with HIV, hepatitis C, and cytomegalovirus, often precede infection-associated ITP. Antibodies develop against viral antigens which exhibit a form of molecular mimicry that leads to cross-reactions with normal platelet antigens.
Anti-body dependant cellular phagocytosis, natural-killer cells dysfunction, complement-dependent cytotoxicity, and cytotoxic T lymphocyte-mediated cytotoxicity can be responsible for destruction of platelets and decreased differentiation of megakaryocytes.
Severe cases of ITP can lead to intracranial hemorrhage and death.
Inherited thrombocytopenia (IT), such as Wiskott-Aldrich syndrome, Alport syndrome and others, is characterized by a severely decreased platelet count, variable platelet size, and abnormal platelet function which can aggravate bleeding. Most forms if IT are caused by rare mutations in genes implicated in megakaryocyte differentiation and/or platelet formation and clearance, although the precise mechanisms remain unclear Jinna & Khandar, StatPearls, Jul 2023. To date, several dozen genes have been implicated in IT, including MYH9, WAS, ANKRD26, RUNX1, and NBEAL2. Common variants in genes like GP6, ITGA2B, and SH2B3 may act as modifiers of disease severity, contributing to inter-individual differences in IT phenotype and mechanisms.
In secondary inherited thrombocytopenia, such as Shwachman–Diamond syndrome and Congenital Amegakaryocytic Thrombocytopenia (CAMT), platelet deficiency arises from bone marrow dysfunction instead of defects in platelet size and function. Pathogenic variants associated with secondary inherited thrombocytopenia, including SBDS, DNAJC21, and EFL1, are mostly inherited in an autosomal recessive manner Nelson et al., GeneReviews, Sep 2024.
CAMT is a rare autosomal recessive disorder with loss of function mutations in MPL gene that codes for thrombopoietin receptor, resulting in severe absence of megakaryocytes and low platelet count from birth.
Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by fever, hemolytic anemia, thrombocytopenia, and renal and neurologic dysfunction. Without treatment, TTP has a mortality of about 90%.
About 95% of TTP cases are acquired via an autoimmune mechanisms against ADAMTS13, a plasma von Willebrand factor (VWF)-cleaving protease primarily synthetized in the liver, in most cases by an unknown trigger Stanley et al., StatPearls, Apr 2023, Santoshi et al., Cureus, Aug 2022. Antiplatelet drugs, immunosuppressive agents, HIV, estrogen-containing birth control, and pregnancy are the most commonly listed triggers for ADAMTS13 autoantibody formation causing acquired TTP.
The hereditary form of TTP (cTTP), also known as Upshaw–Schulman syndrome, is caused by biallelic homozygous or compound heterozygous mutations in the ADAMTS13 gene. Some mutation types are associated with an early onset severe disease form while others may enable residual enzyme activity, producing milder and later-onset forms.
Severe deficiency in ADAMTS13 activity leads to accumulation of ultra-large VWF multimers on the endothelial surface, spontaneous platelet tethering to ultra-large VWF, and thereby to depletion of circulating platelets.
Disseminated intravascular coagulation (DIC), characterized by excessive clot formation (microvascular thrombosis) and bleeding with potentially fatal consequences, is triggered by excessive activation of the clotting cascade, particularly in sepsis, trauma, cancer, and obstetric complications Santoshi et al., Cureus, Aug 2022. The excessive clot formation consumes platelet, fibrinogen, and clotting factors, causing consumption coagulopathy in which the organism is unable to stop the bleeding. When dysregulated, fibrinolysis, that occurs in response to clotting, can become a pathological contributor to bleeding.
Hemolytic uremic syndrome (HUS) is commonly caused Shiga toxin-producing E. coli Bhandari et al., StatPearls, Oct 2023. Additional causes of HUS include autoimmune disorders, genetic mutations, and malignancies. The symptoms of HUS include low platelet counts, hemolytic anemia, and acute kidney failure.
Several mechanisms that underly TP, including thrombosis, vascular occlusion, and microvascular inflammation, are directly responsible for ischemia and organ dysfunction impacting lungs, kidneys, skin, heart, and several other organs.
How similar are human and animal skeletal systems?
This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.
Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance to thrombocytopenia is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.
There are significant differences between mouse and human platelet production and regulation that negatively affect researcher's ability to mimic thrombocytopenia phenotypes, to understand its pathomechanisms, and to develop efficient therapies.
Species-specific differences in platelet clearance and turnover rates
The hormone thrombopoietin (TPO), produced by the liver, is the primary physiological regulator of platelet production. Consequenly, in drug-induced liver injury (DILI), impaired liver function can result in reduction of TPO levels, leading to decreased platelet synthesis.
Mice have a faster platelet turnover rates compared to humans, supported by higher circulating TPO levels and faster clearance rate of TPO compared to humans Kuter, Int. J. Hemat., Jul 2013.
The accelerated platelet production may confer a higher resistance to certain thrombocytopenia-inducing conditions seen in humans.
Species-specific differences in immune system
Platelets are actively monitored by immune cells for signs of senescence, dysfunction, or opsonization (antibody coating).
Macrophages, dendritic cells, neutrophils, and other immune cells, carry the Fcγ receptor (FcyR) that mediates IgG-dependent platelet clearance.
Inter-species and inter-individual differences in polymorphisms and expression of FcR are likely to produce inter-species and inter-patient differences in susceptibility to immune system-mediated thrombocytopenia disorders, and in responses to treatments Liu et al., Blood, Aug 2016, Kaifu & Nakamura, Int. Imm., Jul 2017, Junker et al., Front. Immunol., Jul 2020, Audia et al., Cl. Exp. Imm., Feb 2017.
Species-specific differences in immune system are also likely responsible for false-negative results of toxicity testing in animals and failure of animal testing to protect patients from immune-mediated drug-induced thrombocytopenia Clark & Steger-Hartman, Reg. Tox. & Pharmacol., Jul 2018.
Species-specific differences in platelet receptors
Platelet activation for clot formation is triggered via G-protein coupled protease-activated receptors (PAR), located on the surface of platelets. In humans, the protease thrombin activates PAR1 and PAR4 by cleaving receptors’ extracellular domain.
Unlike human platelets, platelets in mice, rats, guinea pigs, hamsters, and rabbits, do not possess PAR1. Instead they express PAR3 which, unlike PAR1, does not in itself mediate thrombin signaling but acts as a cofactor in tandem with PAR4.
As a result, mouse platelets may respond differently to thrombin than human platelets. Moreover, the absence of PAR1 in rodents makes them poor models for developing and testing PAR1-targeting antithrombotic drugs, such as Vorapaxar Chackalamannil, Med. Chem. Res., Sep 2022.
The hormone TPO mediates its activity through binding to platelets’ transmembrane MPL receptor Kuter & Begley, Blood, Nov 2002.
Histidine at position 499 (H499) in the juxtamembrane domain of MPL, that stabilizes the MPL receptor in an inactive conformation, is specific to humans and to non-human primates Kuter, Int. J. Hemat., Jul 2013.
In mice and rats, the MPL receptor has a higher propensity for dimerization, activation and downstream signaling than the human MPL, altering susceptibility of rodents to MPL-driven diseases, such as congenital amegakaryocytic thrombocytopenia, and responses to MPL-targeted therapies.
Other examples of primate-specific features that contribute to species-specific differences in MPL conformity and activity are threonine at position 496 (T496) and tryptophan at position 491 (W491).
Used in treatment of primary immune thrombocytopenia and aplastic anemia , the TPO receptor agonist Eltrombopag binds to W491 position in MLP receptor, promoting MPL dimerization and activation of JAK2/STAT5 and other pathways to boost platelet production. In mice and rats, W491 is replaced by glutamine (Q491), rendering Eltrombopag inactive, Levy et al., Blood, Mar 2020, underscoring the importance of use of human-based models.
Species-specific differences in platelet central signaling cascade
Factors exogenous to platelets, such as vascular injury, drugs, inflammation, and infection, trigger a coordinated and tightly regulated response of platelet molecular networks known as central signaling cascade. Systems biological pathway analysis-aided multi-omics comparison of central platelet signaling cascades in mice and humans, shows major differences in expression levels and regulatory fine-tuning between the two species, likely to adversely affect translational value of mouse models Balkenhol et al., BMC Gen., Dec 2020.
Species-specific differences in platelet aggregation factors
Wild type rodents do not develop thrombotic thrombocytopenic purpura (TTP), and in ADAMTS13 knock-out mice UL VWF do not accumulate to the same pathological extent as in humans Motto et al., J. Clin. Inv., Oct 2005. This human-specific susceptibility to TTP can be explained by species-specific difference in VWF structure (protein sequence and multimer size), regulation (threshold for VWF release), and clearance mechanisms of VWF (hepatic, enzymatic) that are not well understood.
Species-specific differences in Gb3 distribution
In hemolytic uremic syndrome (HUS), E. Coli-produced Shiga toxins enter the cell by binding to the cell membrane glycolipid globotriaocylceramide (Gb3) via sites on Shiga toxin’s pentameric B subunits.
There are major species-specific differences in cellular Gb3 expression and localization that hinder development of effective treatments for HUS Celi et al., Front. Mol. Biosci., Feb 2022.
In human tissues, including in platelets, heart, kidney, lung, liver, smooth muscle, epithelium of gastrointestinal tract, and neurons, Gb3 is widely distributed.
While in human kidneys Gb3 is abundantly expressed in glomerular endothelial cells, podocytes, mesangial cells, and tubular epithelia, in murine kidneys it is expressed primarily in the proximal tubular epithelial cells.
In contrast to humans and non-human primates, rodents and rabbits develop gastrointestinal and renal tubular epithelial lesions, but fail to develop the glomerular endothelial damage and clinical HUS Hall et al., Toxins, Sep 2017.
Several other mutually interactive human organ systems influence the function of the skeletal system (bone marrow) and can affect susceptibility to thrombocytopenia, including endocrine system (hypothyroidism, steroid-responsive thrombocytopenia, cirrhosis-induced low platelet counts), digestive system (gut microbiome-related inflammation and nutrient malabsorption) and cardiovascular system (congestive heart failure-related platelets splenic sequestration, hypotension-impaired bone marrow perfusion).
Face validity - How well do animal models replicate the human disease phenotype?
Attempts to model human thrombocytopenia in animals have had a varying degree of success over the last six decades of research, depending on the disease (sub)type modeled, the species used, and the method of experimental induction.
Rodent models of primary immune thrombocytopenia (ITP) developed through passive transfer of anti-platelet antiserum or platelet-specific monoclonal antibodies did not recapitulate the autoimmune inflammatory response see in human ITP.
In mouse models of ITP generated by passive transfer of an alloantibody, proinflammatory cytokines that play a critical role in autoimmune inflammation in human ITP, including interleukin-1α/2/6/7/23, granulocyte-macrophage–colony-stimulating factor, monocyte chemoattractant protein, macrophage inflammatory protein, RANTES, tumor necrosis factor-α, and interferon-γ, remain at negligible levels after ITP induction Leontyev et al., Transfusion, May 2014.
The dog model that develops naturally occurring ITP represented more accurately human ITP clinical manifestations than mouse models. However, while it modelled the humoral component of platelet destruction in ITP, it did not fully recapitulate the immunopathogenesis that leads to endogenous autoantibody production, nor did it show direct evidence of autoreactive T cells targeting platelets LeVine et al., Br. J. Haematol., Jul 2014.
Several animal models of inherited thrombocytopenia (IT) were developed by gene editing. One such IT condition is Gray Platelet Syndrome (GPS), characterized by deficiency of platelet α-granules, abnormally large platelets, bleeding, and marrow fibrosis.
The three different mouse strains that were generated by knock-out of GPS-associated NBEAL2 gene recapitulated the GPS platelet phenotype. Nonetheless, in contrast to patient-derived megakaryocytes, the Nbeal2-deficient mouse strains exhibited increase in megakaryocytes number, raising questions about the relevance for humans of GPS mouse models Di Buduo et al., Sci. Rep., Mar 2016.
In an attempt to model the congenital thrombotic thrombocytopenic purpura (cTTP), ADAMTS13 knock-out mice were generated. In contrast to humans with cTTP who often spontaneously develop symptoms early in life, ADAMTS13-deficient mice did not show any symptoms. In this mouse model, features of acute TTP with severe thrombocytopenia were visible only after injection with a strong trigger, such as shiga toxin and recombinant human VWF Motto et al., J. Clin. Inv., Oct 2005, Coppo & Lammle, Haematologica, Apr 2020.
To experimentally induce autoimmune TTP in mice, murine monoclonal antibodies raised in ADAMTS13-deficient mice against murine ADAMTS13 were used. As it was the case for the cTTP mouse model, TTP-like symptoms did not occur in this autoimmune TTP mouse model and could only be induced when recombinant human VWF was injected as an additional trigger.
To obtain a more human-relevant autoimmune TTP animal model, an inhibitory murine anti-human ADAMTS13 monoclonal antibody was administered intravenously to baboons. In contrast to mouse models of TTP, the monoclonal antibody-mediated inhibition of ADAMTS13 in the baboon resulted in severe thrombocytopenia and microangiopathic hemolytic anemia with VWF-rich microthrombi in most organs, without the need for an additional trigger. However, in contrast to humans with TTP, who suffer from renal and neurologic dysfunction with a high mortality rate, none of the non-humane primate TTP models had developed severe organ failure nor had died during the study Coppo & Lammle, Haematologica, Apr 2020.
Drug-induced thrombocytopenia (DIT) modeled in animals by introduction of drug-dependent antibodies isolated from human patients failed to mimic the chronic or relapsing nature of DIT observed in humans. This could be explained by species-specific differences in physiology (platelet turnover, immune system, comorbidities) and differences in acute toxicity testing in animals versus long-term exposure in patients.
A major limitation of rodents in assessing the effect of radiation in radiation-induced thrombocytopenia is that, in humans, radiation follows a heterogenous dose distribution, whereas it penetrates uniformly across much thinner bodies of rodents, leading to overestimation or underestimation of organ-specific effects Singh & Seed, Int. J. Radiat. Bio., Jun 2017.
A variety of experimental induction methods was used to model aplastic anemia (AA) in animals, including injection of allogeneic lymphocytes to mimic autoimmune bone marrow destruction, genetic engineering to replicate hereditary AA types, delivery of drugs like busulfan to induce bone marrow suppression and chemical exposure to agents like benzene to induce bone marrow failure. Models that make use of immune system activators, such as in T-cell-mediated bone marrow destruction, effectively recapitulated pancytopenia characteristic of AA, albeit without capturing the chronic or relapsing nature of AA seen in AA patients Scheinberg & Chen, Sem. Hemat., Apr 2013.
In experimental animal models of disseminated intravascular coagulation (DIC), DIC-like symptoms were induced in mice, rats, and dogs using lipopolysaccharides (LPS), tissue factor, cytokines, and activated factor A-phospholipids infusion. While coagulation activation, platelet consumptions, and microvascular thrombosis, were recapitulated in these DIC animal models, only certain aspects of the full clinical spectrum were captured. With the exception of tissue factor administration, most DIC in vivo models tended to overrepresent thrombotic features and underrepresent fibrinolysis - bleeding diathesis seen in acute promyelocytic leukemia patients Giles et al., J. Clin. Invest., Dec 1984, Suga et al., Int. J. Hemat., Apr 2021. The failure of DIC animal models to capture the full spectrum and clinical heterogeneity of DIC is notably problematic for identification and testing of therapeutic targets, and translation of findings from preclinical research to clinical trials.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
Thrombocytopenia has a multitude of etiologies, often with no identifiable trigger and with overlapping underlying mechanisms.
There is often a disconnect between methods of experimental induction of thrombocytopenia in animals and known etiologies observed in patients.
In addition, animal models of thrombocytopenia do not recapitulate the interindividual heterogeneity in disease mechanisms that arises from interaction of internal and external disease causes.
For example, rodent models of ITP developed through passive transfer of anti-platelet antiserum or platelet-specific monoclonal antibodies did not recapitulate the mechanisms of autoimmune inflammatory response Leontyev et al., Transfusion, May 2014, and there was no evidence that mechanisms of canine ITP were identical to mechanisms of human ITP LeVine et al., Br. J. Haematol., Jul 2014.
In the modeling of AA, the use of benzene to investigate its effect on hematopoiesis mimicked the occupational and environmental exposure in humans Risitano, Clin. Med. & Res., May 2005. Nevertheless, this approach did not take recapitulate immune-mediated mechanisms, viral infections, or hereditary factors. More recently, new mouse models that more closely mimic the human immune-mediated AA phenotype were developed Wang et al., Int. J. Mol. Sci., Feb 2023. However, the mechanisms behind the activation of the immune system in this model might be different from immune-mediated mechanisms in human AA.
In patients with the hereditary form of TTP, over 260 disease-associated ADAMTS13 mutation sites have been identified, involving frameshift, missense, and splice site mutations ClinVar, ADAMTS13. However, the considerable time and cost required to develop transgenic mouse models prohibits investigation in mice of individual and combined effects of such a high number of mutations.
It was reported that in both hereditary Adamts13 knock-out TTP and autoimmune TTP mouse models, TTP-like symptoms could only be induced after injection of an additional trigger Coppo & Lammle, Haematologica, Apr 2020. This suggests that human-specific mechanisms of TTP triggering and progression could be at play, such as in VWF structure, regulation, and clearance mechanisms of VWF.
Gray platelet syndrome (GPS) is a rare inherited thrombocytopenia (IT) caused by mutations in the NBEAL2 gene that lead to disruption of development of alpha granules that stock VWF, fibrinogen, and other proteins involved in clotting. The approach of using Nbeal2-knockout mice reflects the disease etiology since most GPS patients are either homozygous or compound heterozygous for mutations in the NBEAL2 gene. Nevertheless, since the human-specific genetic background is not recapitulated in animal models of IT, the impact of patient-specific modifiers of platelet traits cannot be taken into account.
The mechanisms by which NBEAL2 mutations may lead to immune abnormalities seen in GPS patients remain elusive.
More broadly, inter-species, inter-strain, and inter-individual differences in gene expression regulation and immune system represent a major barrier for understanding the precise mechanisms of IT Di Buduo et al., Sci. Rep., Mar 2016.
Although mice with homozygous knock-out of MPL gene recapitulate CAMT-like features, the underlying mechanisms of hematopoietic stem cells exhaustion in CAMT mouse models remain poorly understood and there is no evidence that pathomechanisms in CAMT mice are of relevance for human patients.
In patients with intravascular coagulation (DIC), the disease mechanisms involve a complex cascade of events, including immune activation, endothelial dysfunction, tissue factor expression, fibrinolysis, and pathogen-host interactions, that are not fully recapitulated in animal models of DIC. For instance, LPS injection and cytokine infusion to mimic sepsis-induced DIC does not replicate pathogen-host interactions. In the same manner, factor Xa and tissue factor administration are ill suited for studying mechanisms of upstream inflammation, immune signaling, and vascular response.
Furthermore, the fact that animal models of DIC do not capture the heterogeneity in DIC etiology represents a major hindrance for developing personalized treatments Toh & Alhamdi, Hemat., Dec 2013.
Predictive validity - How well do animal models predict safety and efficacy of therapies in human patients?
Hematology had a modest 23.9% likelihood of approval from Phase I to Approval over 2011-2020. Hemophilia A and anemia research account for the largest number of transitions within this disease area BIO, Clinical Dev. Success Rates 2011-2020.
First line treatment for primary immune thrombocytopenia (ITP) includes glucocorticoids and intravenous immune globulins that inhibit autoantibody production and platelet degradation.
Plasma infusion is used to treat inherited thrombotic thrombocytopenic purpura (TTP). Glucocorticoids are commonly used together with plasma exchange treatments to treat acquired TTP.
Other drugs used to treat ITP and TTP include monoclonal antibody rituximab, and immunosuppressants vincristine, cyclophosphamide, and cyclosporine A. These are typically adjunctive agents given when first-line therapy fails.
About 80% of patients respond to initial treatment, and the post-treatment mortality is 10 to 15% Jinna & Khandar, StatPearls, Jul 2023, Stanley et al., StatPearls, Apr 2023, Di Buduo et al., eLife, Jun 2021.
For patients with aplastic anemia (AA), allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy is used as the first-line therapy. HSCT was approved in mouse models of AA, however, in patients the risk of graft failure and graft versus host disease after HSCT remain one of the main challenges Wang et al., Int. J. Mol. Sci., Feb 2023.
Importantly, the clinical development of therapies for AA, such as immunosuppressive drugs, did not primarily rely on their success in AA mouse models and were initially investigated in rheumatology and other disorders, underscoring the merit of the human-centric approach and astute observations by clinicians Scheinberg & Chen, Sem. Hemat., Apr 2013.
For patients affected with the severe forms of inherited thrombocytopenia (IT), the treatment of choice is HSCT. Unfortunately, for most patients with this condition transplantation is not recommended as the risks outweigh the benefits.
A significant advance in the treatment of thrombocytopenias is the use of thrombopoietin receptor agonists (TPO-RA), that activate pathways that contribute to an increase in platelet count Kuter, Int. J. Hemat., Jul 2013.
Eltrombopag was approved by the FDA in 2015 for treatment of thrombocytopenia in children one year and older with primary ITP who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. FDA's approval of avatrombopag and lusutrombopag for thrombocytopenia in adults with chronic liver disease followed in 2018. The same year, the FDA had approved romiplostim for pediatric patients with ITP.
However, in patients with IT, platelet response to TPO-RA was variable. Eltrombopag was effective in increasing platelet count in four different forms of IT, which affect more than 55% of patients Zaninetti et al., Haematologica, Mar 2020. Thus, the search for effective drugs to treat all patients with IT continues.
A year later, the FDA had approved caplacizumab for the treatment of TTP in combination with plasma exchange and immunosuppressive therapy. Caplacizumab is a humanized monoclonal antibody fragment that attacks the A1 section of VWF and prevents platelet adhesion FDA, caplacizumab, Mar 2019. However, caplacizumab does not tackle the underlying immune-mediated mechanism of production of autoantibodies against ADAMTS13, requiring immune-suppressive treatments to be used alongside.
Drug-induced thrombocytopenia is caused by poor negative predictive value of animal testing in toxicology Clark & Steger-Hartman, Reg. Tox. & Pharmacol., Jul 2018, Downing et al., JAMA, May 2017.
Chemotherapy-induced thrombocytopenia (CIT) in cancer patients often leads to negative outcomes, and yet, there are no standardized guidelines for preventing or managing CIT. The safety and efficacy of use of TPO-RA for cancer patients remains to be established and so far romiplostim is the only TPO-RA used off label for CIT Al-Samkari, Hematol., Dec 2022, Gao et al., Disc. Oncol., Jan 2023.
In congenital amegakaryocytic thrombocytopenia (CAMT), caused by loss of function MPL mutations, eltrombopag is generally ineffective because functional MPL receptors are absent or severely impaired. Gene therapy to restore MPL function is challenging to execute and species-specific differences in hematopoietic stem cells biology, and immune responses further complicate extrapolation of finding from animal models of CAMT to CAMT patients.
Eltrompobag is not a standard therapy for disseminated intravascular coagulation (DIC), since its mechanism of action does not address the excessive platelet consumption, and its onset is too slow to compensate for the rapid platelets loss that occurs in sepsis, trauma, and other acute conditions. Moreover, by increasing the platelet count, Eltrompobag could potentially worsen microvascular thrombi and produce even more organ damage in DIC patients. Although platelet transfusion, or heparin in certain cases, allow acute management, treatments for DIC that target underlying causes of coagulopathy are missing Pene et al., Ann. Inten. Care, Feb 2025, Costello et al., StatPearls, May 2024.
Currently, there are no available treatments for typical hemolytic uremic syndrome (HUS) and antibiotic therapy is generally avoided, as some antibiotics were found to increase Shiga toxin release Hall et al., Toxins, Sep 2017.
Another challenge relative to repurposed and newly developed therapies for thrombocytopenia is the uncertainty in terms of which patients to treat, with which therapy and for how long Provan & Semple, eBioMed., Jan 2022.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Animal experimentation is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment, and life purpose.
In addition to this baseline, animal experiments inflict severe clinical harm in animals De Vleeschauwer et al., Animals, Aug 2023.
Table S5: Severity classification of chemical disease models
Thrombotic thrombocytopenic purpura - up to severe clinical signs
Table S13: Severity classification of genetically altered (GA) lines
Mortality - severe: GA lines resulting in lethality from 2 weeks post-partum on
Coagulation defects - GA lines with coagulation defects, severity depends on expression of clinical signs
GA lines resulting in long-term moderate pain or short-term severe pain: Severe
GA lines resulting in long-term moderate anxiety or short-term severe anxiety: Severe
Table S6: Severity classification of infectious diseases
Viral and bacterial infection: up to severe clinical signs or long-lasting moderate clinical signs. Sepsis model: severe
Table S3: Severity classification of surgery and surgical induction of disease
Organ/cell transplantation where rejection/failure may lead to severe distress, death or impairment of the general condition of the animal: Severe
Table S8: Severity classification of other disease models
Irradiation ± bone marrow transplantation - Irradiation with a lethal dose with/without reconstitution of the immune system. Irradiation with a (sub)lethal dose with reconstitution with development of GvHD: up to severe clinical signs
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance treatments for human diseases.
However, statistics consistently show that clinical success rates of drugs developed and tested in animals is very low, raising the question of whether it is ethical to put the health of patients at risk.
Over 2011-2020, the overall likelihood of approval of drugs for hematology was higher compared to other disease areas. The Phase I to II transition success rate was 69.6%, well above the 52% average of all indications, while Phase II to III transition success rate was 48.1% versus 28.9% average for all indications BIO, New Clin. Dev. Succ. Rat., 2011-2020.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Animal models do not recapitulate the clinical heterogeneity and inter-patient variability in thrombocytopenia phenotypes, etiologis, and responses to treatments.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, animal research enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible Freedman et al., PLOS Biol., Jun 2015.
Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 Arrive guidelines website.
Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented Percie du Sert et al., BMC Vet. Res., Jul 2020, Bazoit, BioRxiv, Feb 2025.
As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted Yarborough et al., PLOS Biol., Jun 2018.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims Smaldino & McElreath, Roy. Soc. Op. Sci., Sep 2016.
In Summary
Thrombocytopenia (TP) is a hematologic pathology in which counts of platelets are below the lower limit of normal. Patients with thrombocytopenia are at an increased risk of bleeding, thrombosis, and mortality.
It is a clinically heterogenous condition with a multitude of forms, etiologies, and contributing risk factors.
Over the last decades, various TP types were modelled in vivo, causing severe suffering in animals. Yet, owing to species-specific differences in platelet production regulation, receptors, signaling cascade, and immune system, our understanding of underlying mechanisms of this highly heterogenous hematologic condition remains limited.
New therapies are needed to improve survival of patients with disseminated intravascular coagulation, chemotherapy-induced TP, congenital amegakaryocytic TP and inherited TP. Human-based in vitro methods will allow to better predict drug-induced adverse effects and to develop personalized treatments for TP.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Thrombocytopenia
*To model inherited thrombocytopenia (IT), and to investigate the impact of mutations on human thrombopoiesis, using patient-derived megakaryocytes from peripheral blood or bone marrow hematopoietic progenitor cells containing patient-specific genetic backgrounds Di Buduo et al., Sci. Rep., Mar 2016.
*To model secondary inherited thrombocytopenia (SIT), like Shwachman–Diamond Syndrome, and to investigate the effect of genetic mutations on bone marrow failure syndrome. To model drug-induced thrombocytopenia (DIT) and to assess the risk of bone marrow injury induced by chemotherapeutic drugs and radiation, using vascularized human healthy/patient-derived bone marrow-on chip Chou et al., Nature Biomed. Eng., Jan 2020.
*To study the mechanisms by which rare/common variants in IT and SIT affect megakaryocytopoiesis and platelet formation/clearance by gene editing (base editing, overexpression, knock-out) and gene perturbation (CRISP interference, siRNA) experiments in human bone marrow hematopoietic progenitor cells.
*To study the effect of TP-caused vascular occlusion, endothelial injury, and inflammation on organ dysfunction and to identify new therapeutic targets, using immunocompetent multi organs-on-chip/human-on-chip Sasserath et al., Adv. Sci., Jun 2020.
*To investigate the mechanisms of altered platelet generation in inherited (Shwachman–Diamond Syndrome, Fanconi anemia, Dyskeratosis congenita) and acquired (idiopathic aplastic anemia – AA, idiopathic bone marrow failure, radiation/chemotherapy-induced thrombocytopenia – RIT/CIT) bone marrow failure, by using bioengineered 3D human bone marrow niche tissue systems Di Buduo et al., Blood, Apr 2015.
*To dissect the effects of toxic exposure (chemotherapy, drugs, radiation) and viral infections on immune-mediated mechanisms that underly aplastic anemia (AA), drug-induced thrombocytopenia (DIT), secondary immune thrombocytopenia (ITP), and thrombotic thrombocytopenic purpura (TTP), by exposing the culture medium to drugs, human viruses, and other external factors and by measuring change in gene expression (qRT-PCR, RNA-seq), inflammation (cytokine assays), CD8 T cell cytotoxicity, Treg suppression, and other endpoints in immunocompetent microfluidic systems. To determine patients’ susceptibility (immune regulatory genes, HLA alleles etc.) to external risk factors by using cells with patient-specific genetic background.
*To study the epigenetic mechanisms susceptible to regulate immune tolerance, megakaryocytes differentiation, and responses to drugs, infections and other external risk factors in AA, DIT, ITP, TTP, by measuring methylation (bisulfite conversion, pyrosequencing), chromatin remodeling, chromatin immunoprecipitation (ChIP qPCR, ChIP seq), expression of epigenetic regulators (RNA seq) etc., and by analysing the functional effect of epigenetic editing (CRISPR activation/repression, lncRNA) in human hematopoietic progenitor cells.
*To discover new treatments for congenital TTP (cTTP), by identifying recurrent functionally targetable mutations, designing innovative approaches to restore functional ADAMTS13 protein (ASO, CRISPR, vectors of delivery to human cells), using cTTP patient-derived liver cells/liver-on-chip.
*To study the mechanisms of idiopathic primary immune thrombocytopenia (ITP), by exposing human bone marrow hematopoietic progenitor cells to patient-derived T cells, B cells, dendritic cells, macrophages, and antibodies, and by measuring lymphocyte proliferation, cytokine secretion, CD8 T cell-mediated cytotoxicity, B-cell mediated phagocytosis, desialylation and apoptosis, RNA-seq, immune cells metabolomics and proteomics, and other endpoints.
*To investigate the mechanisms of normal and aberrant human hematopoiesis/megakaryocytopoiesis, and to identify new therapeutic targets, by single cell multi-omics analysis in healthy human hematopoietic tissues from early fetal life to adulthood Roy et al., Cell Rep., Sep 2021 and in patient-derived hematopoietic stem and progenitor cells Psaila et al., Mol. Cell, May 2020.
*To assess the effect of drugs on human bone marrow function, using human bone marrow-on-chip containing endosteal and perivascular microenvironments Glaser et al., Biomat., Jan 2022.
*To segment immune thrombocytopenia (ITP) patient populations and to predict responses to immunomodulatory treatments, by studying the effect of FCGR3A 158V/F polymorphism in monocytes and macrophages on IgG binding affinity and phagocytosis rate of opsonized platelets, using patient-derived PBMC-autologous platelets- B cells co-culture systems Audia et al., Cl. Exp. Imm., Feb 2017.
*To examine the role of human Th9 and other T helper cells in primary immune thrombocytopenia (ITP) and to identify human-relevant targets that modulate T helper cell activation, by using PBMC derived from healthy individuals and ITP patients who harbor inborn errors of immunity associated with immune dysregulation, complement deficiency, low immunoglobulin, B cell, and T cells levels Rao et al., J. Allerg. & Clin. Immunol., Apr 2025.
*To study the effect of drug-induced liver injury (DILI) on reduction of thrombopoietin (TPO) levels in humans. To improve prediction of DILI in humans. To investigate eventual overlapping mechanisms in DILI and in drug-induced secondary ITP, by using an immunocompetent human/patient-derived dual bone marrow-liver-on-chip, by exposing the vascular compartment to hepatotoxic drugs, and by measuring biomarkers of hepatoxicity, TPO secretion, platelet output, inflammation, and other endpoints Jiang et al., Arch. Tox., Aug 2023, Lee-Montiel et al., Front. Pharmacol., May 2021.
*To testing safety and efficacy of personalized therapies for inherited thrombocytopenia (IT), using bone marrow-on-chip containing patient-derived iPSC with patient-specific genetic backgrounds Di Buduo et al., eLife, Jun 2021.
*To recapitulate the inter-patient heterogeneity in MPL sequence, conformity, and activity in congenital amegakaryocytic thrombocytopenia (CAMT), by using hematopoietic progenitor cells derived from patients with distinct MPL mutations (ranging from complete loss of function to partial signaling activity), by measuring JAK2-STAT5 signaling (STAT5-responsive luciferase reporters), MPL localisation in response to TPO (immunofluorescence), transcriptional responses to TPO (scRNA seq), platelet output, and other endpoints.
*To develop and test efficacy of innovative therapies tailored for patient-specific MPL mutations types (gene therapy, CRISPR-Cas9, small molecules), in hematopoietic progenitor cells derived from patients with CAMT.
*To model heterogenous etiology of disseminated intravascular coagulation (DIC), to study its mechanisms, and to identify therapeutic targets, by using primary human endothelial cells co-cultured with human PBMC and DIC patient-derived plasma, combined with distinct triggering agents for sepsis-induced, trauma-induced, and malignancy-induced DIC.
*To study the mechanisms that underly Shiga toxin-induced hemolytic uremic syndrome (HUS). To develop and to test treatments for HUS, by using human gut-kidney-on-chip infected with Shiga toxin-producing E. Coli and by measuring cell viability and cell–cell junction integrity Lee et al., Toxins, Nov 2021.
*To recommend the most appropriate stand-alone and combination therapies for each thrombocytopenia type, by assessing safety and efficacy in patient-derived multi organs-on-a-chip in a personalized medicine approach.
Although in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. Complementing in vitro methods with animal
experiments is not effective for human patients, because species-specific differences prevent reliable integration and translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the complete human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!