Blood coagulation abnormalities in multibacillary leprosy patients

Abstract

Background: Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae infection. In 2016, more than 200,000 new cases of leprosy were detected around the world, representing the most frequent cause of infectious irreversible deformities and disabilities.

Principal findings: In the present work, we demonstrate a consistent procoagulant profile on 40 reactional and non-reactional multibacillary leprosy patients. A retrospective analysis in search of signs of coagulation abnormalities among 638 leprosy patients identified 35 leprosy patients (5.48%) which displayed a characteristic lipid-like clot formed between blood clot and serum during serum harvesting, herein named 'leprosum clot'. Most of these patients (n = 16, 45.7%) belonged to the lepromatous leprosy pole of the disease. In addition, formation of the leprosum clot was directly correlated with increased plasma levels of soluble tissue factor and von Willebrand factor. High performance thin layer chromatography demonstrated a high content of neutral lipids in the leprosum clot, and proteomic analysis demonstrated that the leprosum clot presented in these patients is highly enriched in fibrin. Remarkably, differential 2D-proteomics analysis between leprosum clots and control clots identified two proteins present only in leprosy patients clots: complement component 3 and 4 and inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP). In agreement with those observations we demonstrated that M. leprae induces hepatocytes release of IHRP in vitro.

Conclusions: We demonstrated that leprosy MB patients develop a procoagulant status due to high levels of plasmatic fibrinogen, anti-cardiolipin antibodies, von Willebrand factor and soluble tissue factor. We propose that some of these components, fibrinogen for example, presents potential as predictive biomarkers of leprosy reactions, generating tools for earlier diagnosis and treatment of these events.

Fig 1. Multibacillary leprosy patients present prolonged coagulation time in both coagulation pathways.

Coagulation times were determined on plasma from 50 non-leprosy patients (Controls), 9 non-reactional multibacillary patients (MB-NR) and 15 multibacillary erythema nodosum leprosum patients (MB-ENL). A) Activated partial thromboplastin time test (aPPT); B) prothrombin time test (PT). MB-NR group are composed by 10LL and 1 BL patients. MB-ENL group are composed by 13 LL and 1 BL individuals. Neither of them presented the leprosum clot during serum harvesting. All patients’ details are listed in S1 Table. *** indicate p<0.0001 with ANOVA.

Fig 2. Multibacillary leprosy patients present a procoagulant phenotype.

Coagulation parameters were determined on plasma from 50 non-leprosy patients (Controls), 9 non-reactional multibacillary leprosy patients (MB-NR) and 15 multibacillary erythema nodosum leprosum patients (MB-ENL). A) Fibrinogen and B) d-dimer plasma concentrations were determined. C) Platelet counting showed no alteration among samples. MB-NR group are composed by 10LL and 1 BL patients. MB-ENL group are composed by 13 LL and 1 BL individuals. * indicate p<0.01 and *** indicate p<0.0001 with ANOVA.

Fig 3. Leprosum clots formed during serum harvesting of multibacillary patient.

A) Appearance of the leprosum clot during lepromatous patient serum harvesting, after blood coagulation and subsequent centrifugation. The vertical scale bar represents 2 cm and identifies the leprosum clot appearing on the top of normal erythrocyte-rich clot. B) The leprosum clot after PBS washing had a characteristic white-milky color. Scale bar represents 1 cm. C) Representation of a normal blood serum harvesting, after coagulation and subsequent centrifugation. D) Control clot aspect, formed from healthy donors’ plasma by the addition of calcium chloride; E) Leprosum clot distribution among 35 leprosy outpatients (11 females, 24 males; median age, 47.7 years; range, 10–76 years) discriminated by the clinical pole of the disease or presence/absence of reactional episodes (F). G) Vascular abnormalities occurrence in patients who developed leprosum clot during serum harvesting (LC patients) or not (H). MB: multibacillary patients, PB: paucibacillary patients, ENL: erythema nodosum leprosum and T1R: reversal reaction.

Fig 4. Correlation between the leprosum clot and pro-coagulant factors.

Pro-coagulant factors were determined in multibacillary leprosy patient serum presenting the leprosum clot (during LC). We also performed the same analysis in previously harvested serum, varying from weeks to months, from the same group of patients before the occurrence of the leprosum clot (before LC). Levels of anti-cardiolipin IgM (A), soluble tissue factor (B) and von Willebrand factor (C) were determined in serum from 35 leprosy outpatients (11 female, 24 male; mean age, 48 years; range, 10–71 years). 1AU indicates the 1 μg/mL signal of affinity chromatography purified anti-cardiolipin IgM. * indicates p<0.01, ** indicates p<0.005 and *** indicates p<0.001 with ANOVA.

Fig 5. Neutral lipid content in control and leprosum clots.

A) Representative image of a high-performance thin layer chromatography of a leprosum clot (LC) and control clot (CC) showing similarities in their compositions but differences in their amounts of neutral lipids. The lipid standards, S1 and S2, were composed of monoglycerides, diglycerides, and triglycerides (S1) and fatty acids and cholesteryl ester (S2). B) Densitometric analysis of (A) by ImageJ software shows the relative abundance of total neutral lipids in each sample in arbitrary units. Image is representative of four independent experiments. *** indicates p<0.0001 by t test using Mann-Whitney.

Fig 6. Protein-protein interaction network constructed by STRING 10.

(A) Protein differential identification by 2DE in control (CC) and leprosy clots (LC). Pixel intensity value in PPM for each protein. Black bars represent proteins exclusively identified in CC: TPM4 (tropomyosin alpha-4), TMO (tyrosine 3-monooxygenase) KNG1 (kininogen 1), and orange bars represent proteins identified only in LC: C4A (complement component 4A) and IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein). (C) Confidence view based on the Gene Ontology enrichment to biological processes related to blood coagulation (p = 1.469⁻³); (B) Evidence view showing all detected proteins in both clots by different line colors representing the types of association evidence (only associations with p = 1.98⁻¹³ are shown); (C) and inflammatory response (p = 6.369⁻⁴). Stronger associations are represented by thicker lines. IHRP is represented by a small sphere, indicating that there is no crystallographic data about its structure. Proteins highlighted by black rings were exclusively detected in control clots, and proteins highlighted by orange rings were exclusively detected in leprosy clots.

Fig 7. Complement component 4 (C4) and inter-alpha-trypsin inhibitor protein (IHRP) production are induced by M. leprae.

IHRP and C4 were determined in multibacillary leprosy patient serum presenting the leprosum clot (during LC). We also performed the same analysis in previously harvested serum, varying from weeks to months, from the same group of patients before the occurrence of the leprosum clot (before LC). IHRP (A) and C4 (B) are more abundant in leprosy patient sera than healthy controls. Although M. leprae fail to induce C4 synthesis and release by human hepatocytes in vitro (C); we successfully mimetized the phenomenon in vitro observing strong IHRP induction in our model (D). * indicates p<0.05, and ** indicates p<0.001.