A case of acquired transient bleeding diathesis associated with acquired platelet storage pool deficiency and defective thromboxane A2 production

Abstract

Acquired disorders of platelet function are an underdiagnosed cause of bleeding tendency. A 14-year-old girl developed moderate mucocutaneous bleeding two weeks after a Mycoplasma pneumoniae infection successfully treated with clarithromycin. The patient was referred to us 7 months later for laboratory investigation of the persisting bleeding diathesis. The patient’s personal and family histories were negative for bleeding disorders. Complete blood count, von Willebrand Factor levels and coagulation tests were normal; platelet aggregation, ATP secretion, δ-granules content and serum thromboxane B2 levels were defective. At follow-up visits, laboratory parameters and the bleeding diathesis progressively normalized within 2 years. The patient’s condition is compatible with a diagnosis of acquired Storage Pool Deficiency (SPD), associated with defective thromboxane A2 production. To our knowledge, this is the first case of acquired, transient SPD with spontaneous remission. The pathogenic role of Mycoplasma pneumoniae infection or clarithromycin is possible, albeit uncertain.

Keywords:

• Storage Pool Deficiency
• Platelet granules
• ADP
• Platelet aggregation
• Platelet secretion

Introduction

Disorders of platelet function – whether congenital or acquired – are associated with bleeding tendency and are underdiagnosed in clinical practice.¹ The most common congenital platelet function disorders are characterized by impairment of platelet secretion, caused by qualitative/quantitative defects in platelet granules or by abnormalities in signal transduction.¹ The term Storage Pool Deficiency (SPD) is referred to disorders caused by deficiency in platelet granules, which can be selective for alpha (α)- or delta (δ)-granules, or involve both types. Selective deficiency of α-granules has been identified in rare congenital disorders only, such as, for instance, the Gray Platelet Syndrome.¹ The most common congenital abnormality of δ-granules is δ-Storage Pool Deficiency (δ-SPD), characterized by a defect in the number and/or content of δ-granules, which can be syndromic or non-syndromic.² The lack of platelet δ-granules constituents (serotonin, adenine nucleotides, calcium and pyrophosphate) is associated with mucocutaneous bleeding of variable degree, ranging from mild to moderate. Mild thrombocytopenia can be observed in 20–40% of affected patients.³ Abnormalities of platelet aggregation, characterized by lack of amplifying effects by secreted ADP, are of variable severity and may be absent in about 25% of affected patients.⁴ The diagnosis of δ-SPD is based on the measurement of δ-granule constituents, or by inspection of platelets by transmission electron microscopy.⁵ Cases of acquired SPD have been reported, which were most commonly associated with immune or hematological diseases.⁶ Normalization or improvement of the acquired platelet defect never occurred spontaneously, but in association with successful treatment of the underlying disease. Here we report the case of a patient with acquired bleeding diathesis and SPD, associated with defective platelet production of thromboxane A2 (TxA2), temporally associated with Mycoplasma pneumoniae infection and treatment with clarithromycin, which normalized spontaneously within 2 years.

Case description

A 14-year old girl from Sicily was referred to our outpatient clinic in October 2014 for a moderate bleeding diathesis. She had experienced spontaneous bruising, bleeding from minor wounds, and frequent episodes of epistaxis and menorrhagia, which manifested for the first time in March 2014 in the absence of any clinically overt disorders (including immunologic and hematologic diseases), with the exception of a Mycoplasma pneumoniae infection which was diagnosed by means of a serological test 2 weeks before and was successfully treated with clarithromycin 500 mg twice daily for 21 days.

At the time of patient referral, the bleeding assessment tool score of the International Society on Thrombosis and Haemostasis was 6 (normal values for subjects under 18 years of age: 0–2⁷); physical examination revealed a large bluish-purple ecchymosis on the left leg and few smaller resolving bruises on the lower limbs.

The patient’s personal and family histories of bleeding were negative. All parameters of blood count, including the platelet count (208×10⁹/L) and blood cells morphology, were normal. Prothrombin time, activated partial thromboplastin time, von Willebrand factor (vWF) antigen and ristocetin cofactor activity were within their normal ranges. The closure time of the collagen/epinephrine cartridge of Platelet Function Analyzer-100 (PFA-100) was prolonged (>300 seconds; normal range: 76–178 seconds), while that of the collagen/ADP cartridge was normal (93 seconds; normal range: 60–116 seconds), compatible with the presence of a platelet function abnormality and normal vWF activity.⁸ The following parameters of platelet function were investigated: I) platelet aggregation and ATP secretion induced by several agonists, measured simultaneously by lumi-aggregometry (Chrono-log series 560 Havertown, PA, USA) in citrated platelet-rich plasma prepared as previously described,^9,10 according to SCC/ISTH recommendations¹¹; II) platelet content of serotonin, measured by spectrofluorometry,¹² adenosine triphosphate (ATP) and adenosine diphosphate (ADP), measured by luminometry¹³; III) phosphorylation of vasodilator-stimulated phosphoprotein (VASP) induced by prostaglandin E₁ (flow cytometry); IV) serum levels of thromboxane B2 (TxB2, stable metabolite of the platelet agonist TxA2), measured by a selective, competitive enzyme immunoassay (Thromboxane B2 EIA kit, Cayman Chemicals, Ann Arbor, MI, USA).

Platelet aggregation and ATP secretion induced by ADP (2, 4 and 20 µM), epinephrine (10 µM), thrombin receptor activating peptide (TRAP, 10 and 20 µM), collagen (2, 4 and 10 µg/mL) and a thromboxane A₂ analogue (U46619, 1 µM) were partially or severely impaired (Table I, Figure 1). Only platelet aggregation induced by arachidonic acid (1 mM) was within the normal range. The platelet levels of serotonin and ADP were low and the ATP/ADP ratio was high, indicative of defective δ-granules content (Table I). Serum T×B2 levels were lower than normal (45 pmol/10⁸ platelets, normal values 63–472 pmol/10⁸ platelets) (Table I). Phosphorylation of VASP induced by prostaglandin E₁ was normal (PRI: 75%), thus ruling out defects of the P2Y₁₂ receptor for ADP.¹⁴ Platelet aggregation, ATP secretion, ADP and serotonin contents were normal in the patient’s parents. In a follow-up visit 4 months later (February 2015), most platelet parameters normalized or improved, although some of them remained pathological (Table I, Figure 1). Clinically, the hemorrhagic manifestations had improved, and only spontaneous ecchymoses were present. At the next follow-up visit in March 2016 the bleeding manifestations had disappeared and did not relapse thereafter. Subsequent testing in March 2016 showed normalization of platelet function, with δ-granule contents and serum T×B2 levels within their normal ranges, which persisted in two subsequent follow-up visits (Table I, Figure 1).

Figure 1. Platelet aggregation and ATP secretion of citrated PRP from the patient performed during the first visit (October 2014), and subsequent follow-up visits in February 2015 and June 2021, compared to control. Platelets were stimulated with ADP (A), collagen (B) and TRAP (C). Platelet serotonin and adenine nucleotides contents (nmol/10⁸ platelets) at each visit are reported in the table.

Table I. Results of the platelet function tests in the studied patient over time.

	October 2014		February 2015	March 2016	September 2020	June 2021	Normal range
Bleeding diathesis	Spontaneus bruising, bleeding from minor wounds, epistaxis, menorrhagia		Spontaneous bruising	None	None	None
Platelet agonist	Platelet ATP secretion, (nmol/10^8 platelets)
ADP (2 µM)	0		0	0.021	0.746	0.222	0.015 – 1.002
ADP (4 µM)	0		0.08	0.706	–	–	0.040 – 0.815
ADP (20 µM)	0.095		0.245	–	–	–	0.053 – 0.720
Epinephrine (5 µM)	0		0.163	0.811	0.829	0.428	0.039 – 0.972
TRAP (10 µM)	0		0.173	0.969	0.829	0.615	0.027 – 1.356
TRAP (20 µM)	0.056		–	–	–	–	0.478 – 1.239
Collagen (2 µg/mL)	0		0.245	0.930	0.687	0.522	0.281 – 1.090
Collagen (4 µg/mL)	0.044		–	–	–	–	0.237 – 0.988
Collagen (10 µg/mL)	0.117		–	–	–	–	0.321 – 0.976
U46619 (1 µM)	0		0.142	0.838	0.675	0.362	0.069 – 0.952
Arachidonic acid (1 mM)	0.092		0.224	0.680	0.604	0.531	0.104 – 0.891
Platelet agonist	Platelet aggregation, (%)
ADP (2 µM)
maximal	13		28	36	89	78	>21
3 minutes	0		0	36	89	78
ADP (4 µM)
maximal	29		46	74	–	–	>51
3 minutes	2		42	74	–	–
ADP (20 µM)	47		72	–	–	–	>75
Epinephrine (5 µM)	6		75	80	80	87	>10
TRAP (10 µM)	0		56	81	89	96	>21
TRAP (20 µM)
maximal	24		–	–	–	81	>72
3 minutes	17		–	–	–	81
Collagen (2 µg/mL)	0		72	83	91	87	>62
Collagen (4 µg/mL)	5		–	–	–	–	>72
Collagen (10 µg/mL)
maximal	42		–	–	–	–	>73
3 minutes	39		–	–	–	–
U46619 (1 µM)
maximal	27		74	83	89	87	>33
3 minutes	0		74	83	89	87
Arachidonic acid (1 mM)	62		76	79	80	88	>59
	Platelet serotonin and adenine nucleotides content, (nmol/10^8 platelets)
Serotonin	0.14		0.15	0.68	–	–	0.23–0.58
ADP	1.1		1.76	3.29	3.33	3.41	1.99–4.67
ATP	6.5		7.62	8.02	6.22	6.15	5.29–11.34
ATP/ADP ratio	6		4.32	2.43	1.88	1.88	1.30–3.29
Cartridge		PFA-100 closure time, (sec)
ADP/collagen	93		84	79	–	–	60–116
Epinephrine/collagen	>300		234	116	–	–	76–178
		Serum thromboxane B2, (pmol/10^8 platelets)
Serum Thromboxane B2	2.62		39	127	–	–	63–472

Numbers in bold characters indicate pathological results. Abbreviations: ATP= adenosine triphosphate; ADP=adenosine diphosphate; TRAP=thrombin receptor activating peptide; U46619=thromboxane A2 analogue; PFA-100=Platelet Function Analyzer-100. The normal ranges have been derived from testing 33 healthy volunteers in our laboratory.

Discussion

Based on these findings and on the absence of pathological bleeding in the remote patient’s history, we hypothesized that the patient was affected by an acquired, transient form of SPD associated with defective T×A2 production. The platelet aggregation abnormalities of our patient, similarly to those of congenital δ-SPD, are a consequence of defective amplification by secreted ADP.⁹ They are characterized by normal first-wave aggregation in response to adenosine diphosphate (ADP) and epinephrine, diminished or absent second-wave aggregation, and impaired aggregation induced by secretion-inducing agonists, such as collagen and TRAP. Moreover, defective T×A2 production, which contributes with secreted ADP to the amplification of platelet aggregation in normal platelets, could have played an additional role in impairing the aggregation response of our patient’s platelets. Combined defects of δ-granules and T×A2 production have not been described previously in either congenital¹⁵ or acquired forms of SPD, suggesting that the pathogenic mechanism of the acquired defect observed in our patient was not selective for a particular platelet target. The observation that platelet aggregation induced by arachidonic acid (the precursor of TxA2) was normal in our patient, despite partially defective T×A2 production, suggests that either the patient's platelet phospholipase A2 activity was defective or that the amount of T×A2 produced by platelets upon their exposure to high concentrations of exogenous arachidonic acid (1 mM) was sufficient to support normal platelet aggregation. In the absence of alternative potential causes of acquired SPD, we postulate a causal relationship with Mycoplasma pneumoniae infection and/or with the exposure of the patient to clarithromycin. Mycoplasma pneumoniae causes upper and lower respiratory tract infections especially in children and young adults, which may associate with extrapulmonary clinical manifestations, including thrombocytopenia and autoimmune hemolytic anemia.¹⁶ Given that acquired SPD may have an autoimmune genesis in some cases, one plausible hypothesis could be that immunoglobulins generated during Mycoplasma pneumoniae infection or its pharmacological treatment could have caused the platelet defect. Alternative pathogenic mechanisms can be hypothesized, such as direct or indirect toxic effects on the megakaryocytic lineage. Although there have been reports of thrombocytopenia secondary to Mycoplasma pneumoniae¹⁷ or macrolide treatment,¹⁸ no cases of acquired platelet dysfunction have been described so far.

In summary, we report the case of a young girl with acquired bleeding diathesis, normal platelet count and transient severe and complex platelet function defect characterized by acquired SPD and impaired T×A2 production, with spontaneous complete remission. The temporal association with Mycoplasma pneumoniae infection and treatment with clarithromycin could suggest, but not prove, a cause-effect relationship with the acquired platelet defect.

Author contributions statement

M.S., E.A.F., C.G., A.F. and E.B. performed the laboratory analyses. M.S. and B.C. wrote the first draft of the manuscript. M.C. and G.M.P. designed the study, contributed to data interpretation and critically reviewed and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors are thankful to the patient and her family and to the nursing staff of the Hematology Day Hospital of Presidio Ospedaliero San Paolo, ASST Santi Paolo e Carlo, whose cooperation was essential for the collection of samples for this study. The authors acknowledge the support of the APC central fund of the University of Milan.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The raw data supporting the results of this study will be made available upon reasonable request.

Additional information

Funding

The authors received no financial support for the research, authorship, and publication of this article.